Constitutively translocating cell line

ABSTRACT

The present invention relates to agonist-independent methods of screening for compounds that alter GPCR desensitization. Included in the present invention are cell lines containing GRKs, in which GPCRs are desensitized in the absence of agonist; the GRKs may be modified. The present invention relates to methods to determine if a GPCR is expressed at the plasma membrane, and if the GPCR has an affinity for arrestin. Modified GPCRs which have increased arrestin affinity are included in the present invention. These modified GPCRs are useful in methods to screen for compounds that alter desensitization, including both the agonist-independent methods and agonist-dependent methods described herein.

The present application is a Divisional Application of U.S. patentapplication Ser. No. 10/788,197, filed on Feb. 26, 2004, which is aContinuation-In-Part application of International Application No.PCT/US03/14581, filed on May 12, 2003, which claims the benefit of U.S.Provisional Application No. 60/379,986 filed on May 13, 2002; and U.S.Provisional Application No. 60/401,698 filed on Aug. 7, 2002; which arehereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to methods of assaying GPCRdesensitization in a agonist-independent manner, host cells useful insuch methods, methods of the identification of compounds that alter GPCRdesensitization, the compounds identified, and their use in diseasetreatment.

BACKGROUND

G protein-coupled receptors (GPCRs) are cell surface proteins thattranslate hormone or ligand binding into intracellular signals. GPCRsare found in all animals, insects, and plants. GPCR signaling plays apivotal role in regulating various physiological functions includingphototransduction, olfaction, neurotransmission, vascular tone, cardiacoutput, digestion, pain, and fluid and electrolyte balance. Althoughthey are involved in numerous physiological functions, GPCRs share anumber of common structural features. They contain seven membranedomains bridged by alternating intracellular and extracellular loops andan intracellular carboxyl-terminal tail of variable length.

GPCRs have been implicated in a number of disease states, including, butnot limited to; cardiac indications such as angina pectoris, essentialhypertension, myocardial infarction, supraventricular and ventriculararrhythmias, congestive heart failure, atherosclerosis, renal failure,diabetes, respiratory indications such as asthma, chronic bronchitis,bronchospasm, emphysema, airway obstruction, upper respiratoryindications such as rhinitis, seasonal allergies, inflammatory disease,inflammation in response to injury, rheumatoid arthritis, chronicinflammatory bowel disease, glaucoma, hypergastrinemia, gastrointestinalindications such as acid/peptic disorder, erosive esophagitis,gastrointestinal hypersecretion, mastocytosis, gastrointestinal reflux,peptic ulcer, Zollinger-Ellison syndrome, pain, obesity, bulimianervosa, depression, obsessive-compulsive disorder, organ malformations(for example, cardiac malformations), neurodegenerative diseases such asParkinson's Disease and Alzheimer's Disease, multiple sclerosis,Epstein-Barr infection and cancer.

The magnitude of the physiological responses controlled by GPCRs islinked to the balance between GPCR signaling and signal termination. Thesignaling of GPCRs is controlled by a family of intracellular proteinscalled arresting. Arrestins bind activated GPCRs, including those thathave been agonist-activated and especially those that have beenphosphorylated by G protein-coupled receptor kinases (GRKs).

Receptors, including GPCRs, have historically been targets for drugdiscovery and therapeutic agents because they bind ligands, hormones,and drugs with high specificity. Approximately fifty percent of thetherapeutic drugs in use today target or interact directly with GPCRs.See e.g., Jurgen Drews, (2000) “Drug Discovery: A HistoricalPerspective,” Science 287:1960-1964.

There is a need for accurate, easy to interpret methods of detecting Gprotein-coupled receptor activity and methods of assaying GPCR activity.One method, as disclosed in Barak et al., U.S. Pat. Nos. 5,891,646 and6,110,693, uses a cell expressing a GPCR and a conjugate of an arrestinand a detectable molecule, the contents of which are incorporated byreference in their entirety.

Although only several hundred human GPCRs are known, it is estimatedthat upwards of a thousand GPCRs exist in the human genome. Of theseknown GPCRs, many are orphan receptors that have yet to be associatedwith a ligand.

The majority of the existing methods for identifying GPCR antagonistsare dependent on the presence of agonist. Assays for identifyingcompounds that prevent the activation of GPCRs typically require thatthe GPCR is first activated in order to identify interfering compounds.For receptors with known agonists, these agonists are currently used toactivate these receptors. However, many GPCRs are orphan receptors withno known ligand or agonist.

The agonist-dependence of GPCR assays continues to be a problem becauseantagonist discovery for orphan receptors is typically dependent on theprior discovery of agonist or ligand. Agonist-independent methods toscreen for compounds that alter GPCR desensitization will (1) eliminatethe step of agonist-addition in screening methods, and (2) enableidentification of compounds that alter the desensitization of orphanreceptors. Agonist-independent methods will eliminate the step ofidentifying an agonist of an orphan receptor prior to screening forcompounds that alter desensitization of the orphan receptor.

SUMMARY

The present invention relates to methods of identifying compounds whichalter GPCR internalization.

A first aspect of the present invention is a method of identifying acompound which alters GPCR internalization, including: (a) providing acell including a GPCR, an arrestin, and a modified GRK, wherein saidGPCR is at least partially internalized in an agonist-independent mannerupon expression of said GRK; (b) exposing said cell to the compound(s);(c) determining the cellular distribution of the GPCR, arrestin, ormodified GRK; and (d) monitoring a difference between (1) thedistribution of the GPCR, arrestin, or modified GRK in the cell in thepresence of the compound(s) and (2) the distribution of the GPCR,arrestin, or modified GRK in the cell in the absence of the compound(s).An agonist may not be provided in the above method. In the method, adifference between (1) and (2) of step (d) may indicate modulation ofGPCR internalization.

The GRK may be over-expressed, its expression may be inducible, and itmay include a CAAX motif. The GRK may be GRK1, GRK2, GRK3, GRK4, GRK5,GRK6, or a biologically active fragment thereof.

The GPCR may be modified to have enhanced phosphorylation by a GRK. TheGPCR may be β₂AR(Y326A), a GPCR listed in FIG. 1A-1C, an orphan GPCR, amodified GPCR, a taste receptor, a Class A GPCR, a Class B GPCR, amutant GPCR, or a biologically active fragment thereof.

The arrestin may be visual arrestin, cone arrestin, β-arrestin 1,β-arrestin-2, or a biologically active fragment thereof.

The GPCR, GRK, or arrestin may be detectably labeled. A moleculeinvolved in desensitization may be detectably labeled, or a moleculethat interacts with a molecule involved in desensitization may bedetectably labeled.

In a further aspect, the present invention relates to a method ofidentifying a compound that alters GPCR phosphorylation, including: (a)providing a cell including a GPCR and a GRK; (b) exposing the cell tothe compound(s); and (c) determining whether GRK phosphorylation of theGPCR is altered in the presence of the compound(s).

The cellular distribution of the GPCR or GRK may be determined. Adifference may be monitored between (1) the distribution of the GPCR orGRK in the cell in the presence of the compound(s) and (2) thedistribution of the GPCR or GRK in the cell in the absence of thecompound(s). A difference may be correlated between (1) and (2) to thephosphorylation of the GPCR.

The GRK may not be located in the plasma membrane, indicating that GRKphosphorylation of the GPCR is altered. The phosphorylation state of theGPCR may be determined. The activity of the GRK may be determined. Theability of the GPCR to be internalized may be determined.

In an additional aspect, the present invention relates to a method ofdetermining if a GPCR is expressed at the plasma membrane, including:(a) providing a cell including a GPCR, an arrestin, and a GRK, whereinthe arrestin is detectably labeled; (b) determining the cellulardistribution of the arrestin; and (c) correlating the cellulardistribution of the arrestin to the ability of the GPCR to be expressedat the plasma membrane. The arrestin may be localized in vesicles, pitsendosomes, or elsewhere in the desensitization pathway.

Additionally, the present invention relates to a further method ofdetermining if a GPCR is expressed at the plasma membrane, including:(a) providing a cell including a GPCR and a GRK, wherein the GRK isdetectably labeled; (b) determining the cellular distribution of theGRK, and (c) correlating the cellular distribution of the GRK to theability of the GPCR to be expressed at the plasma membrane. The GRK maybe localized at the plasma membrane.

In a further aspect, the present invention relates to a method ofanalyzing the ability of a GPCR to bind arrestin, including: (a)providing a cell including a GPCR, an arrestin, and a GRK, wherein thearrestin is detectably labeled; (b) determining the cellulardistribution of the arrestin; and (c) correlating the cellulardistribution of the arrestin to the ability of the GPCR to bindarrestin. The arrestin or the GPCR may be localized in vesicles, pits,or endosomes.

In an additional aspect, the present invention relates to a compoundidentified by a method of the present invention.

In a further aspect, the present invention is related to a method oftreating a disease by modulating desensitization of a GPCR in a hostcell, including: (a) providing a compound identified by a method of thepresent invention; and (b) administering the compound to a host.

Another aspect of the invention relates to a host cell including a GPCRand a modified GRK. The GRK may be inducible or over-expressed. The hostcell may further include arrestin, wherein the arrestin may bedetectably labeled. The GPCR, GRK or another molecule involved indesensitization, or a molecule that interacts with a molecule involvedin desensitization may be detectably labeled.

A further aspect of the present invention relates to a method ofmodifying a nucleic acid encoding a GRK in which a GPCR isconstitutively internalized, including: (a) providing a nucleic acidencoding a GRK; (b) mutating the nucleic acid encoding a GRK such thatthe encoded GRK includes a CAAX motif, wherein the modified GRKphosphorylates a GPCR in the absence of agonist; and (c) expressing themodified GRK in a cell. The nucleic acid encoding a GRK may include SEQID No: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, or34.

The present invention also relates to a kit for identifying a compoundthat modulates the internalization of a GPCR, including a host cellincluding a GPCR and a modified GRK.

In a further aspect, the present invention relates to a modified GPCRincluding a NPXXY motif, and a carboxyl terminal tail, wherein thecarboxyl terminal tail includes a putative site of palmitoylation andone or more clusters of phosphorylation, wherein the carboxyl terminaltail includes a retained portion of a carboxyl-terminus region of afirst GPCR portion fused to a portion of a carboxyl-terminus from asecond GPCR, and wherein the second GPCR includes the one or moreclusters of phosphorylation and further includes a second putative siteof palmitoylation approximately 10 to 25 amino acid residues downstreamof a second NPXXY motif. The first GPCR may be a Class A receptor. Thefirst GPCR may be hGPR3, hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, orhGPR22. The second GPCR may be a Class B receptor. The Class B receptormay be selected from the group consisting of a vasopressin V2 receptor,a neurotensin-1 receptor, a substance P receptor, and an oxytocinreceptor.

The present invention relates to a nucleic acid encoding a modifiedGPCR. Included in the present invention are nucleic acids selected fromthe group consisting of SEQ ID Nos: 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,and 90. Also included in the present invention are expression vectorsincluding the nucleic acid. Host cells including the expression vectoror the nucleic acid are also included.

In a further aspect, the present invention relates to a method ofscreening compounds for GPCR activity including the steps of: (a)providing a cell that expresses at least one modified GPCR, wherein thecell further includes arrestin conjugated to a detectable molecule; b)exposing the cell to the compound; (c) detecting location of thearrestin within the cell; (d) comparing the location of the arrestinwithin the cell in the presence of the compound to the location of thearrestin within the cell in the absence of the compound; and (e)correlating a difference between (1) the location of the arrestin withinthe cell in the presence of the compound and (2) the location of thearrestin within the cell in the absence of the compound. The arrestinmay be detected in endosomes, endocytic vesicles, or pits.

A further aspect of the present invention is a kit for identifying amolecule that modulates the activity of a GPCR, including a cell thatexpresses at least one modified GPCR, wherein the cell further includesa molecule involved in desensitization conjugated to a detectablemolecule.

BRIEF DESCRIPTION OF DRAWINGS

The objects and advantages of the invention will be understood byreading the following detailed description in conjunction with thedrawings in which:

FIG. 1A-1C is a list of GPCRs that may be used with the presentinvention.

FIG. 2A-2Q is a list of GRKs that may be used with the presentinvention. Amino acid and nucleic acid sequences of certain GRKs areshown. The amino acid and nucleic acid sequences of GRK2-C20, a modifiedGRK, are shown.

FIG. 3A-3BB is a list of GPCRs that have been modified to have enhancedaffinity for arrestin. The amino acid and nucleic acid sequences areshown.

FIG. 4 illustrates the agonist-independent translocation of arrestin-GFPto GPCRs in the presence of GRK2-C20.

FIG. 5 illustrates the agonist-independent translocation of arrestin-GFPto GPCRs in the presence of GRK2-C20.

FIG. 6 illustrates the agonist-independent translocation of arrestin-GFPto GPCRs in the presence of GRK2-C20.

FIG. 7 illustrates the agonist-independent translocation of arrestin-GFPto GPCRs in the presence of GRK2-C20.

FIG. 8 demonstrates that losartan, a nonpeptide antagonist/inverseagonist of the AT1AR, inhibits the ligand-independent translocation ofarrestinGFP to the AT1AR induced by expression of GRK2-C20. Data plottedare the mean±SD for a representative experiment performed in triplicate.

FIG. 9 illustrates the percent inhibition of constitutive, GRK2-C20induced, arrestinGFP translocation to the AT1AR by losartan treatmentfor 3 hours.

FIG. 10 illustrates the percent inhibition of constitutive, GRK2-C20induced, arrestinGFP translocation to the AT1AR by losartan treatmentfor 18 hours.

DETAILED DESCRIPTION

In accordance with the present invention there may be employedconventional molecular biology, microbiology, immunology, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Sambrook et al,“Molecular Cloning; A Laboratory Manual” (3^(rd) edition, 2001);“Current Protocols in Molecular Biology” Volumes I-IV [Ausubel, R. M.,ed. (2002 and updated bimonthly)]; “Cell Biology: A Laboratory Handbook”Volumes I-III [J. E. Celis, ed. (1994)]; “Current Protocols inImmunology” Volumes I-IV [Coligan, J. E., ed. (2002 and updatedbimonthly)]; “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “NucleicAcid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)];“Transcription And Translation” [B. D. Hames & S. J. Higgins, eds.(1984)]; “Culture of Animal Cells, 4^(th) edition” [R. I. Freshney, ed.(2000)]; “Immobilized Cells And Enzymes” [IRL Press, (1986)]; B. Perbal,“A Practical Guide To Molecular Cloning” (1988); Using Antibodies: ALaboratory Manual. Portable Protocol No. I, Harlow, Ed and Lane, David(Cold Spring Harbor Press, 1998); Using Antibodies: A Laboratory Manual,Harlow, Ed and Lane, David (Cold Spring Harbor Press, 1999); “GProtein-Coupled Receptors” [T. Haga, et al., eds. (1999)].

Unless otherwise stated, the following terms used in the specificationand claims have the meanings given below:

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An “origin of replication” refers to those DNA sequences thatparticipate in the initiation of DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

The expression of a coding sequence in a host cell may be inducible. Byinducible, it is meant that the expression can be regulated. Forexample, the nucleic acid may be present in the cell, but it is notexpressed until a necessary signal is provided. Typically, inducibleexpression of a protein is controlled by a promoter that requires anecessary signal to induce transcription of the protein. However,expression may also be induced by a process or sequence that increasesthe number of DNA sequences of interest in the cell. Such processes orsequences include origins of replication, as well as the physicaladdition of DNA to a cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined by mapping with nuclease S1), as well as protein binding domains(consensus sequences) responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequencesin addition to the −10 and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

The term “oligonucleotide,” as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target DNA sequence. This means thatthe primers must be sufficiently complementary to hybridize with theirrespective strands. Therefore, the primer sequence need not reflect theexact sequence of the template. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the primer, withthe remainder of the primer sequence being complementary to the strand.Alternatively, non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer sequence hassufficient complementarity with the sequence of the strand to hybridizetherewith and thereby form the template for the synthesis of theextension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into chromosomal DNA making up thegenome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

Two DNA sequences are “substantially homologous” when at least about 65%(preferably at least about 80%, and most preferably at least about 90 or95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

It should be appreciated that also within the scope of the presentinvention are DNA sequences encoding the same amino acid sequence as SEQID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,71, 73, 75, 77, 79, 81, 83, 85, 87, and 89, but also those which aredegenerate to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, and 90. By“degenerate to” is meant that a different three-letter codon is used tospecify a particular amino acid.

“Arrestin” means all types of naturally occurring and engineeredvariants of arrestin, including, but not limited to, visual arrestin(sometimes referred to as Arrestin 1), cone arrestin (sometimes referredto as arrestin-4), β-arrestin 1 (sometimes referred to as Arrestin 2),and β-arrestin 2 (sometimes referred to as Arrestin 3).

“βARK1” is a GRK termed β-adrenergic receptor kinase 1, also calledGRK2.

“βAR” is a GPCR termed a β-adrenergic receptor.

“Internalization” of a GPCR is the translocation of a GPCR from the cellsurface membrane to an intracellular vesicular membrane, where it may beinaccessible to substances remaining outside the cell.

“Carboxyl-terminal tail” means the carboxyl-terminal tail of a GPCRfollowing membrane span 7. The carboxyl-terminal tail of many GPCRsbegins shortly after the conserved NPXXY motif that marks the end of theseventh transmembrane domain (i.e. what follows the NPXXY motif is thecarboxyl-terminal tail of the GPCR). The carboxyl-terminal tail may berelatively long (approximately tens to hundreds of amino acids),relatively short (approximately tens of amino acids), or virtuallynon-existent (less than approximately ten amino acids). As used herein,“carboxyl-terminal tail” shall mean all three variants (whetherrelatively long, relatively short, or virtually non-existent), and mayor may not contain palmitoylated cysteine residue(s).

“Class A receptors” preferably do not translocate together with arrestinproteins to endocytic vesicles or endosomes in association witharrestin-GFP in HEK-293 cells.

“Class B receptors” preferably do translocate together with arrestinproteins to endocytic vesicles or endosomes associated with arrestin-GFPin HEK-293 cells.

“DACs” mean any desensitization active compounds. Desensitization activecompounds are any compounds that influence the GPCR desensitizationmechanism by either stimulating or inhibiting the process. DACs mayinfluence the GPCR desensitization pathway by acting on any cellularcomponent of the process, as well as any cellular structure implicatedin the process, including but not limited to: arrestins, GRKs, GPCRs,phosphoinositide 3-kinase, AP-2 protein, clathrin, protein phosphatases,and the like. DACs may include, but are not limited to, compounds thatinhibit arrestin translocating to a GPCR, compounds that inhibitarrestin binding to a GPCR, compounds that stimulate arrestintranslocating to a GPCR, compounds that stimulate arrestin binding to aGPCR, compounds that inhibit GRK phosphorylation of a GPCR, compoundsthat stimulate GRK phosphorylation of a GPCR, compounds that stimulateor inhibit GRK binding to a GPCR, compounds that inhibit proteinphosphatase dephosphorylation of a GPCR, compounds that stimulateprotein phosphatase dephosphorylation of a GPCR, compounds that preventGPCR internalization or recycling to the cell surface, compounds thatregulate the release of arrestin from a GPCR, antagonists of a GPCR,inverse agonists and the like. DACs may inhibit or stimulate the GPCRdesensitization process and may not bind to the same ligand binding siteof the GPCR as traditional agonists and antagonists of the GPCR. DACsmay act independently of the GPCR, i.e., they do not have highspecificity for one particular GPCR or one particular type of GPCRs.DACs may bind the same site(s) as agonist or antagonist but do notdesensitize the receptor (perhaps by not altering the receptor to beproperly phosphorylated or bind to arrestin or any other protein). DACsmay bind to allosteric sites on the receptor and inhibit or enhancedesensitization.

“Detectable molecule” means any molecule capable of detection byspectroscopic, photochemical, biochemical, immunochemical, electrical,radioactive, and optical means, including but not limited to,fluorescence, phosphorescence, and bioluminescence and radioactivedecay. Detectable molecules include, but are not limited to, GFP,luciferase, β-galactosidase, rhodamine-conjugated antibody, and thelike. Detectable molecules include radioisotopes, epitope tags, affinitylabels, enzymes, fluorescent groups, chemiluminescent groups, and thelike. Detectable molecules include molecules which are directly orindirectly detected as a function of their interaction with othermolecule(s).

“GFP” means Green Fluorescent Protein which refers to various naturallyoccurring forms of GFP which may be isolated from natural sources orgenetically engineered, as well as artificially modified GFPs. GFPs arewell known in the art. See, for example, U.S. Pat. Nos. 5,625,048;5,777,079; and 6,066,476. It is well understood in the art that GFP isreadily interchangeable with other fluorescent proteins, isolated fromnatural sources or genetically engineered, including but not limited to,yellow fluorescent proteins (YFP), red fluorescent proteins (RFP), cyanfluorescent proteins (CFP), blue fluorescent proteins, luciferin, UVexcitable fluorescent proteins, or any wave-length in between. As usedherein, “GFP” shall mean all fluorescent proteins known in the art.

“Unknown or Orphan Receptor” means a GPCR whose ligands are unknown.

“Downstream” means toward a carboxyl-terminus of an amino acid sequence,with respect to the amino-terminus.

“Upstream” means toward an amino-terminus of an amino acid sequence,with respect to the carboxyl-terminus.

Amino acid substitutions may also be introduced to substitute an aminoacid with a particularly preferable property. For example, a Cys may beintroduced a potential site in order to allow formation of disulfidebridges with another Cys. A His may be introduced as a particularly“catalytic” residue (i.e., His can act as an acid or base and is themost common amino acid in biochemical catalysis). Pro may be introducedbecause of its particularly planar structure, which induces β-turns inthe protein's structure.

Two amino acid sequences are “substantially homologous” when at leastabout 70% of the amino acid residues (preferably at least about 80%, andmost preferably at least about 90 or 95%) are identical, or representconservative substitutions.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to prevent, and preferably reduce some feature ofpathology such as for example, elevated blood pressure, respiratoryoutput, etc.

A DNA sequence is “operatively linked” to an expression control sequencewhen the expression control sequence controls and regulates thetranscription and translation of that DNA sequence. The term“operatively linked” includes having an appropriate start signal (e.g.,ATG) in front of the DNA sequence to be expressed and maintaining thecorrect reading frame to permit expression of the DNA sequence under thecontrol of the expression control sequence and production of the desiredproduct encoded by the DNA sequence. If a gene that one desires toinsert into a recombinant DNA molecule does not contain an appropriatestart signal, such a start signal can be inserted in front of the gene.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleoside or nucleotide bases. For example, adenine (A) and thymine (T)are complementary nucleobases that pair through the formation ofhydrogen bonds.

The term “standard hybridization conditions” refers to salt andtemperature conditions substantially equivalent to 5×SSC and 65° C. forboth hybridization and wash. However, one skilled in the art willappreciate that such “standard hybridization conditions” are dependenton particular conditions including the concentration of sodium andmagnesium in the buffer, nucleotide sequence length and concentration,percent mismatch, percent formamide, and the like. Also important in thedetermination of “standard hybridization conditions” is whether the twosequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standardhybridization conditions are easily determined by one skilled in the artaccording to well known formulae, wherein hybridization is typically10-20° C. below the predicted or determined Tm with washes of higherstringency, if desired.

By “animal” is meant any member of the animal kingdom includingvertebrates (e.g., frogs, salamanders, chickens, or horses) andinvertebrates (e.g., worms, etc.). “Animal” is also meant to include“mammals.” Preferred mammals include livestock animals (e.g., ungulates,such as cattle, buffalo, horses, sheep, pigs and goats), as well asrodents (e.g., mice, hamsters, rats and guinea pigs), canines, felines,primates, lupine, camelid, cervidae, rodent, avian and ichthyes.

“Antagonist(s)” include all agents that interfere with wild-type and/ormodified GPCR binding to an agonist, wild-type and/or modified GPCRdesensitization, wild-type and/or modified GPCR binding arrestin,wild-type and/or modified GPCR endosomal localization, internalization,and the like, including agents that affect the wild-type and/or modifiedGPCRs as well as agents that affect other proteins involved in wild-typeand/or modified GPCR signaling, desensitization, endosomal localization,resensitization, and the like.

“Modified GPCR” means a GPCR that has one or more modifications in theamino acid sequence of its carboxyl-terminal tail. As such, thecarboxyl-terminal tail may be modified in whole or in part. Thesemodifications in the amino acid sequence include mutations of one ormore amino acids, insertion of one or more amino acids, deletion of oneor more amino acids, and substitutions of one or more amino acids inwhich one or more amino acids are deleted and one or more amino acidsare added in place of the deleted amino acids. Such modified GPCRs aredescribed herein, as well as in U.S. Ser. No. 09/993,844, which isincorporated herein by reference in its entirety.

“GPCR” means G protein-coupled receptor and includes GPCRs naturallyoccurring in nature, as well as GPCRs which have been modified.

“Putative site of palmitoylation” means an expected site of palmitateaddition, preferably a cysteine residue. In the GPCRs used in thepresent invention, the putative site of palmitoylation is preferably 10to 25, preferably 15 to 20, amino acid residues downstream of the NPXXYmotif.

“Clusters of phosphorylation sites” mean clusters of amino acid residuesthat may be efficiently phosphorylated and thus readily function asphosphorylation sites. The clusters of amino acids occupy two out oftwo, two out of three, three out of three positions, three out of fourpositions, four out of four, four out of five positions, five out offive, and the like consecutive amino acid positions in the carboxylterminal tail of a GPCR. These clusters of phosphorylation sites arepreferably clusters of serine (S) and/or threonine (T) residues.Clusters of phosphorylation sites may be substituted, inserted, or addedon to a GPCR sequence so that the resulting modified GPCR binds arrestinwith sufficient affinity to recruit arrestin into endosomes.

“NPXXY motif” means a conserved amino acid motif that marks the end ofthe seventh transmembrane domain. The conserved amino acid motif beginsmost frequently with asparagine and proline followed by two unspecifiedamino acids and then a tyrosine. The two unspecified amino acids mayvary among GPCRs but the overall NPXXY motif is conserved.

“Abnormal GPCR desensitization” and “abnormal desensitization” mean thatthe GPCR desensitization pathway is disrupted such that the balancebetween active receptor and desensitized receptor is altered withrespect to wild-type conditions. Either there is more active receptorthan normal or there is more desensitized receptor than wild-typeconditions. Abnormal GPCR desensitization may be the result of a GPCRthat is constitutively active or constitutively desensitized, leading toan increase above normal in the signaling of that receptor or a decreasebelow normal in the signaling of that receptor.

“Biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject; wherein said sample can be blood, serum, aurine sample, a fecal sample, a tumor sample, a cellular wash, an oralsample, sputum, biological fluid, a tissue extract, freshly harvestedcells, or cells which have been incubated in tissue culture.

“Concurrent administration,” “administration in combination,”“simultaneous administration,” or “administered simultaneously” meanthat the compounds are administered at the same point in time orsufficiently close in time that the results observed are essentially thesame as if the two or more compounds were administered at the same pointin time.

“Conserved abnormality” means an abnormality in the GPCR pathway,including but not limited to, abnormalities in GPCRs, GRKs, arresting,AP-2 protein, clathrin, protein phosphatase and the like, that may causeabnormal GPCR signaling. This abnormal GPCR signaling may contribute toa GPCR-related disease.

“Desensitized GPCR” means a GPCR that presently does not have ability torespond to agonist and activate conventional G protein signaling.

“Desensitization pathway” means any cellular component of thedesensitization process, as well as any cellular structure implicated inthe desensitization process and subsequent processes, including but notlimited to, arresting, GRKs, GPCRs, AP-2 protein, clathrin, proteinphosphatases, and the like. In the methods of assaying of the presentinvention, the polypeptides may be detected, for example, in thecytoplasm, at a cell membrane, in clathrin-coated pits, in endocyticvesicles, endosomes, any stages in between, and the like.

“GPCR signaling” means GPCR induced activation of G proteins. This mayresult in, for example, cAMP production.

“G protein-coupled receptor kinase” (GRK) includes any kinase that hasthe ability to phosphorylate a GPCR. Certain GRKs which may be used inthe present invention are listed in FIG. 2A-2Q. Splice variants,biologically active fragments, modified GRKs, and GRKs from animals andother organisms are included.

“Homo sapiens GPCR” means a naturally occurring GPCR in a Homo sapiens.

“Inverse agonist” means a compound that, upon binding to the GPCR,inhibits the basal intrinsic activity of the GPCR. An inverse agonist isa type of antagonist.

“Modified GRK” means a GRK modified such that it alters desensitization.

“Naturally occurring GPCR” means a GPCR that is present in nature.

“Odorant ligand” means a ligand compound that, upon binding to areceptor, leads to the perception of an odor including a syntheticcompound and/or recombinantly produced compound including agonist andantagonist molecules.

“Odorant receptor” means a receptor protein normally found on thesurface of olfactory neurons which, when activated (normally by bindingan odorant ligand) leads to the perception of an odor.

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Sensitized GPCR” means a GPCR that presently has ability to respond toagonist and activate conventional G protein signaling.

“Modulation” includes at least an up-regulation or down-regulation ofthe expression, or an increase or decrease in activity of a protein.Modulation of a protein includes the up-regulation, down-regulation,increase or decrease in activity of a protein or compound that regulatesa protein. Modulation also includes the regulation of the gene, themRNA, or any other step in the synthesis of the protein of interest.

An “overexpressed” protein refers to a protein that is expressed atlevels greater than wild-type expression levels.

“Modified GRK” means a GRK that has one or more modifications in theamino acid sequence at the C-terminus of the GRK. The modified GRKconstitutively localizes to the plasma membrane. Preferably, the GRK ismodified by the addition of a CAAX motif.

“CAAX” (SEQ ID NO: 95) motif means a four amino acid sequence, wherein Cis cysteine; A is an aliphatic amino acid; and X is the C-terminal aminoacid of the protein.

A “constitutive” activity means an activity that occurs in the absenceof agonist. For example, the modified GRK constitutively localizes tothe plasma membrane means that the modified GRK localizes to the plasmamembrane in the absence of agonist.

“GRK-C20” refers to a modified GRK which has the ability to have ageranylgeranyl group added to it. GRK2-C20 is a GRK2 modified in thismanner. Preferably, the GRK-C20 contains a CAAX motif.

The present inventors developed an agonist-independent method to screenfor compounds that alter GPCR desensitization. They developed cell linesin which GPCRs are desensitized in the absence of agonist. These celllines include GRKs, which may be modified. Using these cell lines, theydeveloped methods to screen for compounds that alter GPCRdesensitization in the absence of agonist. These methods eliminate thestep of agonist addition from the screening method. The elimination ofthis step (1) creates more efficient screening methods for compoundsthat alter desensitization of GPCRs with known agonists, and (2)provides screening methods for compounds that alter desensitization oforphan GPCRs, which have no known agonist. They developed methods todetermine if a GPCR is expressed at the plasma membrane, and determineif the GPCR has an affinity for arrestin; preferably these methodsutilize an orphan GPCR and host cells containing a GRK, wherein the GPCRis at least partially internalized in an agonist-independent manner uponexpression of the GRK, thus eliminating the need for agonist addition.They modified GPCRs to increase their affinities for arrestin. Thesemodified GPCRs are useful in the agonist-independent methods to screenfor compounds that alter desensitization.

GPCRs and Desensitization

The exposure of a GPCR to agonist produces rapid attenuation of itssignaling ability that involves uncoupling of the receptor from itscognate heterotrimeric G-protein. The cellular mechanism mediatingagonist-specific or homologous desensitization is a two-step process inwhich agonist-occupied receptors are phosphorylated by a Gprotein-coupled receptor kinases (GRKs) and then bind an arrestinprotein.

It is known that after agonists bind GPCRs, G-protein coupled receptorkinases (GRKs) phosphorylate intracellular domains of GPCRs. Afterphosphorylation, an arrestin protein associates with theGRK-phosphorylated receptor and uncouples the receptor from its cognateG protein. The interaction of the arrestin with the phosphorylated GPCRterminates GPCR signaling and produces a non-signaling, desensitizedreceptor.

The arrestin bound to the desensitized GPCR targets the GPCR toclathrin-coated pits or other cellular machinery for endocytosis (i.e.,internalization) by functioning as an adaptor protein, which links theGPCR to components of the endocytic machinery, such as adaptor protein-2(AP-2) and clathrin. The internalized GPCRs are dephosphorylated and arerecycled back to the cell surface resensitized, or are retained withinthe cell and degraded. The stability of the interaction of arrestin withthe GPCR is one factor that dictates the rate of GPCR dephosphorylation,recycling, and resensitization. The involvement of GPCR phosphorylationand dephosphorylation in the desensitization process has beenexemplified in U.S. Ser. No. 09/933,844, filed Nov. 5, 2001, thedisclosure of which is hereby incorporated by reference in its entirety.

Using methods described herein, the present inventors identified certainGPCRs which do not have an affinity for arrestin. They modified theseGPCRs to comprise one or more sites of phosphorylation, preferablyclusters of phosphorylation sites, properly positioned in theircarboxyl-terminal tail. This modification allows the modified GPCR toform a stable complex with an arrestin that will internalize as a unitinto endosomes. These modified GPCRs may be useful in methods ofassaying GPCR activity. These modified GPCRs may be useful to identifyagonists of the GPCRs. These modified GPCRs may be useful in theagonist-independent screening methods described herein.

Agonist-independent screening methods using GPCRs altered to contain aDRY motif are described in U.S. Ser. No. 10/054,616, which isincorporated herein by reference in its entirety. The alteration of theGPCR is included in that screening process; each GPCR to be utilizedmust be altered in that manner.

The present inventors developed agonist-independent screening methodsusing GRKs, which may be modified. These GRKs phosphorylate GPCRs in theabsence of agonist. These phosphorylated GPCRs internalize in theabsence of agonist. The present inventors developed agonist-independentmethods of screening for antagonists of GPCR internalization utilizingthese modified GRKs. These methods do not require the GPCR alterationsdescribed in U.S. Ser. No. 10/054,616.

Previously, certain GRKs were shown to constitutively localize in theplasma membrane. Inglese et al constructed GRK2-C20 which wasconstitutively isoprenylated and localized to the membranes.

The present inventors determined that cellular expression of GRKs thatconstitutively localize in the plasma membrane results in constitutivedesensitization of GPCRs. These GRKs may be over-expressed, theirexpression may be inducible, the nucleic acids encoding them may belocated in a vector or integrated into the genome. The present inventorsconstructed host cell expressing a GRK that constitutively localizes inthe plasma membrane. These host cells may also express arrestin. Tothese host cells, they introduced a GPCR of interest. Using theGRK-containing cells, they developed methods to determine if a GPCR ofinterest is expressed at the plasma membrane, analyze the ability of aGPCR to bind arrestin, and detect constitutively desensitized GPCRs.They built upon these desensitization methods and developedagonist-independent methods of identifying compounds that alter GPCRdesensitization. These methods are useful for the identification ofcompounds that alter the internalization of GPCRs, whether the GPCRagonist is known or unknown.

The present inventors also determined that increased expression ofwild-type or modified GRKs increased desensitization, irrespective ofwhether the GRK constitutively localized in the plasma membrane.

The present invention is related to modified GPCRs, polypeptides ofmodified GPCRs, nucleic acid molecules that encode the modified GPCRs,vectors containing the nucleic acid molecules which encode the modifiedGPCRs, vectors enabling the nucleic acid construction of the modifiedGPCRs, and cells containing modified GPCRs. The invention furtherrelates to assay systems using the modified GPCRs, assay systems usingthe cells containing modified GPCRs, compounds identified using theassay systems, methods of treatment using the compounds identified,methods of disease diagnosis using the assay systems, and kitscontaining assay reagents of the present invention and cells of thepresent invention.

Mutations can be made in the GPCR or modified GPCR such that aparticular codon is changed to a codon which codes for a different aminoacid. Such a mutation is generally made by making the fewest nucleotidechanges possible. A substitution mutation of this sort can be made tochange an amino acid in the resulting protein in a non-conservativemanner (i.e., by changing the codon from an amino acid belonging to agrouping of amino acids having a particular size or characteristic to anamino acid belonging to another grouping) or in a conservative manner(i.e., by changing the codon from an amino acid belonging to a groupingof amino acids having a particular size or characteristic to an aminoacid belonging to the same grouping). Such a conservative changegenerally leads to less change in the structure and function of theresulting protein. A non-conservative change is more likely to alter thestructure, activity or function of the resulting protein. The presentinvention should be considered to include sequences containingconservative changes which do not significantly alter the activity orbinding characteristics of the resulting protein.

In a particular embodiment, the modified GPCRs of the present inventioninclude GPCRs that have been modified to have one or more sites ofphosphorylation, preferably clusters of phosphorylation sites, properlypositioned in its carboxyl-terminal tail. These modified GPCRs recruitarrestin to endosomes within approximately 30 minutes of agoniststimulation. These modified GPCRs recruit arrestin to endosomes in thecells described herein, in which the GPCR is phosphorylated in anagonist-independent manner.

The modified GPCRs of the present invention comprise one or more sitesof phosphorylation, preferably one or more clusters of phosphorylationsites, properly positioned in its carboxyl-terminal tail. The presentinventors have discovered that GPCRs containing one or more sites ofphosphorylation, preferably clusters of phosphorylation sites, properlypositioned in its carboxyl-terminal tail have an increased affinity forarrestin and colocalize with arrestin in endosomes upon GPCRphosphorylation, either after stimulation with agonist or in anagonist-independent manner as described herein. The present inventorshave also discovered that the one or more sites of phosphorylation,preferably clusters of phosphorylation sites, must be optimallypositioned within the GPCR tail for the GPCR to have an increasedaffinity for arrestin. Therefore, the modified GPCRs may be constructedsuch that the one or more sites of phosphorylation, preferably clustersof phosphorylation sites, are optimally positioned within thecarboxyl-terminal tail. The portions of polypeptides, which are to befused together to form the modified GPCR, are chosen such that the oneor more sites of phosphorylation, preferably clusters of phosphorylationsites, are reliably positioned properly within the carboxyl-terminaltail. In the alternative, the location of discrete point mutations tocreate the modified GPCR may be chosen so that the one or more sites ofphosphorylation, preferably clusters of phosphorylation sites, areproperly positioned within the carboxyl-terminal tail.

The present inventors have discovered that the modified GPCRs of thepresent invention are useful in assays for screening compounds that mayalter G protein-coupled receptor (GPCR) activity. Examples of assays inwhich the present invention may be used include, but are not limited to,those as described in U.S. Pat. Nos. 5,891,646 and 6,110,693, thedisclosures of which are hereby incorporated by reference in theirentireties. Additional examples of assays in which the present inventionmay be used include, but are not limited to, assays using FluorescentResonance Energy Transfer (FRET) and assays using BioluminescenceResonance Energy Transfer (BRET) technology as described in Angers, S.,Salahpour, A., Joly, E., Hilairet, S., Chelsky, “β₂-adrenergic receptordimerization in living cells using bioluminescence resonance energytransfer (BRET),” Proc. Natl, Acad. Sci. USA 97, 7: 3684-3689.

The present inventors have determined that these modified GPCRs areuseful in agonist-independent assays for screening compounds that mayalter GPCR internalization. Examples of assays in which the presentinvention may be used include, but are not limited to, assays describedherein.

Methods of Enhancing GPCR Desensitization

Provided in the present invention are methods of enhancing GPCRdesensitization. One embodiment is related to the expression of GRKs,which may be modified. The GRKs may be over-expressed or theirexpression may be inducible. These methods may be used to analyze thedesensitization of a GPCR, including a modified GPCR, an orphan GPCR, ataste receptor, a mutant GPCR, the β2AR Y326A GPCR mutant, or anotherGPCR. Certain GPCRs useful in the present invention are listed in FIG.1A-1C.

In a preferred embodiment, a cell is provided that contains anexpression system and a nucleic acid encoding a GRK. The GRK may bemodified such that the expression of the GRK results in constitutivedesensitization of the GPCR. The GRK may be over-expressed and itsexpression may be inducible.

Preferably, host cells are provided which include a GRK, which may bemodified, and arrestin. A GPCR is then added to these cells. Theagonist-independent desensitization of the GPCR is detected. FIGS. 4, 5,6, and 7 are examples of this method. Detection methods are describedbelow.

The present invention provides methods of determining if the GPCR ofinterest is expressed at the plasma membrane. GPCRs expressed at theplasma membrane are useful in the previously mentioned methods ofcompound identification.

A preferred method of determining if a GPCR of interest is expressed atthe plasma membrane includes: (a) providing a cell including a GPCR, anarrestin, and a GRK, wherein the arrestin is detectably labeled; (b)determining the cellular distribution of the arrestin; and (c)correlating the cellular distribution of the arrestin to the ability ofthe GPCR to be expressed at the plasma membrane.

Preferred embodiments of this aspect of the invention are described inExamples 2, 3, 4, 5, 6, and 7 and illustrated in FIGS. 4, 5, 6, and 7.

Another method of determining if a GPCR of interest is expressed at theplasma membrane includes: (a) providing a cell comprising a GPCR and aGRK, wherein the GRK is detectably labeled; (b) determining the cellulardistribution of the GRK; and (c) correlating the cellular distributionof the GRK to the ability of the GPCR to be expressed at the plasmamembrane.

The present invention provides methods of analyzing the ability of aGPCR to bind arrestin. GPCRs which bind arrestin are useful in thepreviously mentioned methods of compound identification.

A preferred method of analyzing the ability of a GPCR to bind arrestinincludes: (a) providing a cell including a GPCR, an arrestin, and a GRK,wherein the arrestin is detectably labeled; (b) determining the cellulardistribution of the arrestin; and (c) correlating the cellulardistribution of the arrestin to the ability of the GPCR to bindarrestin.

Preferred embodiments of this aspect of the invention are described inExamples 2, 3, 4, 5, 6, and 7, and illustrated in FIGS. 4, 5, 6, and 7.

Using this method, certain GPCRs will bind arrestin and desensitize.However, certain GPCRs will not desensitize without modification of theGPCR, as described in U.S. Ser. No. 09/993,844. The present inventorsmodified several GPCRs, including known and orphan GPCRs, listed in FIG.3A-3BB. Upon modification, these modified GPCRs constitutivelydesensitized in the above system.

Modified GPCRs

The present invention is related to modified GPCRs. Modified GPCRs ofthe present invention may comprise one or more modifications in theircarboxyl-terminal tail. These modifications may comprise inserting oneor more sites of phosphorylation, preferably clusters of phosphorylationsites, within certain regions of the carboxyl-terminal tail. As such,the carboxyl-terminal tail may be modified in whole or in part. Thecarboxyl-terminal tail of many GPCRs begins shortly after a conservedNPXXY motif that marks the end of the seventh transmembrane domain (i.e.what follows the NPXXY motif is the carboxyl-terminal tail of the GPCR).The carboxyl-terminal tail of many GPCRs comprises a putative site ofpalmitoylation approximately 10 to 25 amino acid residues, preferably 15to 20 amino acid residues, downstream of the NPXXY motif. This site istypically one or more cysteine residues. The carboxyl-terminal tail of aGPCR may be relatively long, relatively short, or virtuallynon-existent. The present inventors have determined that thecarboxyl-terminal tail of a GPCR determines the affinity of arrestinbinding.

The present inventors have discovered that specific amino acid motifs inthe carboxyl-terminal tail promote formation of a stable GPCR/arrestincomplex and thus ultimately may promote recruitment of arrestin toendosomes. These amino acid motifs comprise one or more amino acids,preferably clusters of amino acid residues, that may be efficientlyphosphorylated and thus readily function as phosphorylation sites. Theclusters of amino acids may occupy two out of two, two out of three,three out of three, three out of four positions, four out of four, fourout of five positions, five out of five, and the like consecutive aminoacid positions. Accordingly, the clusters of amino acids that promoteformation of a stable GPCR/arrestin complex are “clusters ofphosphorylation sites,” These clusters of phosphorylation sites arepreferably clusters of serine and threonine residues.

GPCRs that form stable complexes with arrestin comprise one or moresites of phosphorylation, preferably clusters of phosphorylation sites.In addition to the presence of the one or more sites of phosphorylation,preferably clusters of phosphorylation sites, it has been discoveredthat the sites must be properly positioned within the carboxyl-terminaltail to promote formation of a stable GPCR/arrestin complex. To promoteformation of a stable GPCR/arrestin complex, the one or more sites ofphosphorylation, preferably one or more clusters of phosphorylation, maybe approximately 15 to 35 (preferably 15 to 25) amino acid residuesdownstream of a putative site of palmitoylation of the GPCR. Inaddition, the one or more sites of phosphorylation, preferably one ormore clusters of phosphorylation, may be approximately 20 to 55(preferably 30 to 45) amino acid residues downstream of the NPXXY motifof the GPCR. GPCRs containing one or more sites of phosphorylation,preferably clusters of phosphorylation sites, properly positioned aretypically Class B receptors.

By way of example, it has been discovered that the V2R receptorcomprises a cluster of phosphorylation sites (SSS) that promotesformation of a stable GPCR/arrestin complex at 19 amino acid residuesdownstream of the putative site of palmitoylation and 36 amino acidresidues downstream of the NPXXY motif. The NTR-2 receptor comprises acluster of phosphorylation sites (STS) that promotes formation of astable GPCR/arrestin complex at 26 amino acid residues downstream of theputative site of palmitoylation and 45 amino acid residues downstream ofthe NPXXY motif. The oxytocin receptor (OTR) receptor comprises twoclusters of phosphorylation sites (SSLST and STLS) that promoteformation of a stable GPCR/arrestin complex, one at 20 amino acidresidues downstream of the putative site of palmitoylation and the otherat 29 amino acid residues downstream of the putative site ofpalmitoylation, and one at 38 amino acid residues downstream of theNPXXY motif and the other at 47 amino acid residues downstream of theNPXXY motif, respectively. The substance P receptor (SPR, also known asthe neurokinin-1 receptor) comprises a cluster of phosphorylation sites(TTIST) that promotes formation of a stable GPCR/arrestin complex at 32amino acid residues downstream of the putative site of palmitoylationand 50 amino acid residues downstream of the NPXXY motif.

The present inventors have determined that GPCRs that lack one or moresites of phosphorylation, preferably clusters of phosphorylation,properly positioned within the carboxyl terminal tail form GPCR/arrestincomplexes that are less stable and dissociate at or near the plasmamembrane. These GPCRs are typically Class A receptors, olfactoryreceptors, taste receptors, and the like. However, the present inventorshave discovered that stable GPCR/arrestin complexes may be achieved withGPCRs naturally lacking one or more sites of phosphorylation and havinga lower affinity for arrestin by modifying the carboxyl-terminal tailsof these receptors. Preferably, the carboxyl-terminal tails are modifiedto include one or more sites of phosphorylation, preferably one or moreclusters of phosphorylation sites, properly positioned within thecarboxyl terminal tail.

The present invention includes the polypeptide sequences of thesemodified GPCRs. The modified GPCRs of the present invention includeGPCRs that have been modified to have one or more sites ofphosphorylation, preferably one or more clusters of phosphorylation,properly positioned in their carboxyl terminal tails. The polypeptidesequences of the modified GPCRs of the present invention also includesequences having one or more additions, deletions, substitutions, ormutations. These mutations are preferably substitution mutations made ina conservative manner (i.e., by changing the codon from an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic to an amino acid belonging to the same grouping). Such aconservative change generally leads to less change in the structure andfunction of the resulting protein. The present invention should beconsidered to include sequences containing conservative changes which donot significantly alter the activity or binding characteristics of theresulting protein.

The modified GPCRs of the present invention include GPCRs containing aNPXXY motif a putative site of palmitoylation approximately 10 to 25amino acid residues (preferably 15 to 20 amino acids) downstream of theNPXXY motif, and a modified carboxyl-terminal tail. The modifiedcarboxyl-terminal tail has one or more sites of phosphorylation,preferably one or more clusters of phosphorylation sites, such that thephosphorylation sites are approximately 15 to 35, preferably 15 to 25,amino acid residues downstream of the putative site of palmitoylation ofthe modified GPCR. The modified carboxyl-terminal tail may have one ormore sites of phosphorylation, preferably one or more clusters ofphosphorylation sites, such that the phosphorylation sites areapproximately 20 to 55, preferably 30 to 45, amino acid residuesdownstream of the NPXXY of the modified GPCR.

The present invention further includes isolated nucleic acid moleculesthat encode modified GPCRs. It should be appreciated that also withinthe scope of the present invention are DNA sequences encoding modifiedGPCRs which code for a modified GPCR having the same amino acid sequenceas the modified GPCRs, but which are degenerate. By “degenerate to” itis meant that a different three-letter codon is used to specify aparticular amino acid.

As one of skill in the art would readily understand, the carboxyl-tailof many GPCRs may be identified by the conserved NPXXY motif that marksthe end of the seventh transmembrane domain.

To create a modified GPCR containing a modified carboxyl-terminus regionaccording to the present invention, a GPCR lacking phosphorylation sitesor clusters of phosphorylation sites or with a lower or unknown affinityfor arrestin may have one or more additions, substitutions, deletions,or mutations of amino acid residues in its carboxyl-terminal tail. Theseadditions, substitutions, deletions, or mutations are performed suchthat the carboxyl-terminal tail is modified to comprise one or moresites of phosphorylation, preferably clusters of phosphorylation sites.By way of example, discrete point mutations of the amino acid residuesmay be made to provide a modified GPCR. By way of example threeconsecutive amino acids may be mutated to serine residues to provide amodified GPCR. These mutations are made such that the one or more sitesof phosphorylation, preferably clusters of phosphorylation sites, areproperly positioned within the carboxyl terminal tail.

In addition, to create a modified GPCR containing a modifiedcarboxyl-terminal tail region, mutations may be made in a nucleic acidsequence of a GPCR lacking sites of phosphorylation or clusters ofphosphorylation sites or with a lower or unknown affinity for arrestinsuch that a particular codon is changed to a codon which codes for adifferent amino acid, preferably a serine or threonine. Such a mutationis generally made by making the fewest nucleotide changes possible. Asubstitution mutation of this sort can be made to change an amino acidin the resulting protein to create one or more sites of phosphorylation,preferably clusters of phosphorylation sites. Also by way of example,discrete point mutations of the nucleic acid sequence may be made. Thephosphorylation sites are positioned such that they are locatedapproximately 15 to 35 amino acid residues downstream of the putativesite of palmitoylation of the modified GPCR.

Furthermore, to provide modified GPCRs of the present invention, a GPCRlacking properly positioned phosphorylation sites or with a lower orunknown affinity for arrestin may also have its carboxyl-terminal tail,in whole or in part, exchanged with that of a GPCR having properlypositioned clusters of phosphorylation sites. The site of exchange maybe after or including the conserved NPXXY motif. As an alternative, aputative site of palmitoylation of a GPCR may be identified atapproximately 10 to 25 (preferably 15 to 20) amino acid residuesdownstream of the conserved NPXXY motif, and the site of exchange may beafter or including the palmitoylated cysteine(s). Preferably, thecarboxyl-terminal tail of a GPCR lacking properly positioned clusters ofphosphorylation sites or with a lower or unknown affinity for arrestinis exchanged at an amino acid residue in close proximity to a putativesite a palmitoylation. More preferably, the carboxyl-terminal tail of aGPCR lacking properly positioned clusters of phosphorylation sites orwith a lower or unknown affinity for arrestin is exchanged at a putativesite of palmitoylation approximately 10 to 25 (preferably 15 to 20)amino acid residues downstream of the NPXXY motif, such that thepalmitoylated cysteine residue is maintained. Exchanging in thepreferred manner allows the clusters of phosphorylation sites to bereliably positioned properly within the carboxyl-terminal tail of themodified GPCR. The tails may be exchanged and the modified GPCRs may beconstructed accordingly by manipulation of the nucleic acid sequence orthe corresponding amino acid sequence.

In a further alternative, the carboxyl-tail of a GPCR, for example aGPCR not containing the NPXXY motif, may be predicted from ahydrophobicity plot and the site of exchange may be selectedaccordingly. Based on a hydrophobicity plot, one of skill in the art maypredict a site where it is expected that the GPCR may anchor in themembrane and then predict where to introduce a putative site ofpalmitoylation accordingly. Using this technique GPCRs having neither aNPXXY motif nor a putative site of palmitoylation may be modified tocreate a point of reference (e.g. a putative site of palmitoylation).The introduced putative site of palmitoylation may then be used toposition a tail exchange.

The carboxyl-terminal tail used for the exchange may be from a secondGPCR having one or more properly positioned clusters of phosphorylationsites and having a putative site of palmitoylation approximately 10 to25 (preferably 15 to 20) amino acid residues downstream of a NPXXYmotif. The tail as identified may be exchanged, after or including theconserved NPXXY motif. As an alternative, a putative site ofpalmitoylation of a GPCR may be identified at approximately 10 to 25(preferably 15 to 20) amino acid residues downstream of the conservedNPXXY motif and the tail may be exchanged after or including thepalmitoylated cysteine(s). Preferably, the carboxyl-terminal tail of aGPCR having clusters of phosphorylation sites is exchanged at an aminoacid residue in close proximity to a putative site of palmitoylation.More preferably, the carboxyl-terminal tail of a GPCR having clusters ofphosphorylation sites is exchanged at a putative site of palmitoylationapproximately 10 to 25 (preferably 15 to 20) amino acid residuesdownstream of the NPXXY motif, such that the portion of thecarboxyl-terminal tail containing the clusters of phosphorylation sitesbegins at the amino acid residue immediately downstream of thepalmitoylated cysteine residue. Exchanging in the preferred mannerallows the clusters of phosphorylation sites to be reliably positionedproperly within the carboxyl-terminal tail of the modified GPCR. Thecarboxyl-terminal tail having clusters of phosphorylation sites used forthe exchange may have a detectable molecule conjugated to thecarboxyl-terminus. The tails may be exchanged and the modified GPCRs maybe constructed accordingly by manipulation of the nucleic acid sequenceor the corresponding amino acid sequence.

In addition, the carboxyl-terminal tail portion used for the exchangemay originate from a polypeptide synthesized to have an amino acidsequence corresponding to an amino acid sequence from a GPCR having oneor more sites of phosphorylation, preferably one or more clusters ofphosphorylation sites. The synthesized polypeptide may have a putativesite of palmitoylation approximately 10 to 25 (preferably 15 to 20)amino acid residues downstream of a NPXXY motif. The synthesizedpolypeptide may have one or more additions, substitutions, mutations, ordeletions of amino acid residues that does not affect or alter theoverall structure and function of the polypeptide.

Furthermore, the carboxyl-terminal tail portion used for the exchangemay originate from a naturally occurring polypeptide recognized to havean amino acid sequence corresponding to an amino acid sequence from aGPCR having one or more clusters of phosphorylation sites. Thepolypeptide may have a putative site of palmitoylation approximately 10to 25 (preferably 15 to 20) amino acid residues downstream of a NPXXYmotif. The polypeptide may have one or more additions, substitutions,mutations, or deletions of amino acid residues that does not affect oralter the overall structure and function of the polypeptide.

A modified GPCR containing a modified carboxyl-terminus region may becreated by fusing a first carboxyl-terminal tail portion of a GPCRlacking properly positioned clusters of phosphorylation sites or with alower or unknown affinity for arrestin with a second carboxyl-terminaltail portion of a GPCR or polypeptide having one or more clusters ofphosphorylation sites. The second GPCR or polypeptide used for theexchange may have a putative site of palmitoylation approximately 10 to25 (preferably 15 to 20) amino acid residues downstream of a NPXXYmotif. Accordingly, the modified carboxyl-terminus region of themodified GPCR comprises a portion of a carboxyl-terminal tail from aGPCR lacking properly positioned clusters of phosphorylation sites orwith a lower or unknown affinity for arrestin fused to a portion of acarboxyl-terminal tail of a GPCR or polypeptide having clusters ofphosphorylation sites. The tail of a GPCR lacking properly positionedclusters of phosphorylation sites may be exchanged after or includingthe conserved NPXXY motif, and fused to a carboxyl-terminal tailcontaining clusters of phosphorylation sites, after or including theconserved NPXXY motif. As an alternative, the tail of a GPCR lackingproperly positioned clusters of phosphorylation sites may be exchangedafter or including the palmitoylated cysteine(s), and fused to a tailcontaining clusters of phosphorylation sites, after or including thepalmitoylated cysteine(s). The tails may be exchanged and the modifiedGPCRs may be constructed accordingly by manipulation of the nucleic acidsequence or the corresponding amino acid sequence.

In a further alternative, the carboxyl-tail of a GPCR, for example aGPCR not containing the NPXXY motif, may be predicted from ahydrophobicity plot and exchanged accordingly. The site of exchange maybe selected according to the hydrophobicity plot. Based on ahydrophobicity plot, one of skill in the art may predict a site where itis expected that the GPCR may anchor in the membrane and then predictwhere to introduce a putative site of palmitoylation accordingly. Usingthis technique GPCRs having neither a NPXXY motif nor a putative site ofpalmitoylation may be modified to create a point of reference (e.g. aputative site of palmitoylation). The introduced putative site ofpalmitoylation may be then used to position a tail exchange. Afterintroduction of a putative site of palmitoylation, the resulting tailmay be fused with a second carboxyl-terminal tail portion of a GPCR orpolypeptide having one or more clusters of phosphorylation sites andhaving a putative site of palmitoylation approximately 10 to 25(preferably 15 to 20) amino acid residues downstream of a NPXXY motif.

Preferably, the modified carboxyl-terminus region of the modified GPCRis fused at amino acid residues in close proximity to a putative site ofpalmitoylation. More preferably, the modified carboxyl-terminus regionof the modified GPCR is fused such that the portion from the first GPCRwith a lower affinity for arrestin comprises amino acid residues fromthe NPXXY motif through a putative site of palmitoylation approximately10 to 25 (preferably 15 to 20) amino acid residues downstream of theNPXXY motif and the portion from the second GPCR having clusters ofphosphorylation sites and a putative site of palmitoylationapproximately 10 to 25 (preferably 15 to 20) amino acid residuesdownstream of a NPXXY motif comprises amino acid residues beginning withan amino acid residue immediately downstream of the putative site ofpalmitoylation of the second GPCR extending to the end of thecarboxyl-terminus. This fusion is preferred because the clusters ofphosphorylation sites are reliably positioned properly within thecarboxyl-terminal tail and the modified GPCR maintains its structure andability to function.

By way of example, a Class A receptor or an orphan receptor may have aportion of its carboxyl-terminal tail exchanged with a portion of acarboxyl-terminal tail from a known Class B receptor. Further, receptorshaving virtually non-existent carboxyl-terminal tails, for example,olfactory receptors and taste receptors, may have a portion of theircarboxyl-terminal tails exchanged with a portion of a carboxyl-terminaltail from a known Class B receptor. The Class B receptor tail used forthese exchanges may have a detectable molecule fused to thecarboxyl-terminus.

Modified GPCRs may be generated by molecular biological techniquesstandard in the genetic engineering art, including but not limited to,polymerase chain reaction (PCR), restriction enzymes, expressionvectors, plasmids, and the like. By way of example, vectors, such as apEArrB (enhanced arrestin binding), may be designed to enhance theaffinity of a GPCR lacking clusters of phosphorylation sites forarrestin. To form a vector, such as a pEArrB vector, PCR amplified DNAfragments of a GPCR carboxyl-terminus, which forms stable complexes witharrestin, may be digested by appropriate restriction enzymes and clonedinto a plasmid. A schematic of one such plasmid is illustrated in FIG.4A. The DNA of a GPCR, which is to be modified, may also be PCRamplified, digested by restriction enzymes at an appropriate location,and subcloned into the vector, such as pEArrB, as illustrated in FIG.4B. When expressed, the modified GPCR will contain a polypeptide fusedto the carboxyl-terminus. The polypeptide will comprise clusters ofphosphorylation sites. Preferably, the polypeptide originates from theGPCR carboxyl-terminus of a receptor that forms stable complexes witharrestin.

Such modified GPCRs may also occur naturally as the result of aberrantgene splicing or single nucleotide polymorphisms. Such naturallyoccurring modified GPCRs would be predicted to have modified endocytictargeting. These naturally occurring modified GPCRs may be implicated ina number of GPCR-related disease states.

As shown in FIG. 3A-3BB, the present inventors modified several GPCRs.The β2-adrenergic receptor, dopamine D1A receptor, mu opiod receptor,orphan GPR3, orphan GPR6, orphan GPR12, orphan GPR7, orphan GPR8, orphanGPR55, orphan SREB2, and orphan SREB3 were modified as described herein.These modified GPCRs contain a properly positioned V2R cluster ofphosphorylation sites (SSS) within the modified GPCR's tail.

As may be shown by standard receptor binding assays, the modifiedreceptors are essentially indistinguishable from their wild-typecounterparts except for an increased affinity for arrestin and thus anincreased stability of their complex with arrestin and in their abilityto traffic with arrestin and in their ability to recycle andresensitize. For example, the modified receptors are appropriatelyexpressed at the membrane and possess similar affinity for agonists orligands. However, the modified GPCRs have an increased affinity forarrestin and thus form a more stable complex with arrestin than theirwild-type counterparts and may remain bound to arrestin when traffickingto endosomes.

These modified GPCRs are useful in assays to screen for an agonist ofthe GPCR, as well as in agonist-independent assays to identify compoundsthat alter GPCR desensitization.

Methods of Assaying GPCR Activity Using the Modified GPCRs

The modified GPCRs of the present invention are useful in methods ofassaying GPCR activity. The modified GPCRs of the present invention maybe used in assays to study GPCRs that have weaker than desiredinteractions or associations with arrestins and GPCRs that have unknowninteractions or associations with arresting. Methods of the presentinvention that use the modified GPCRs provide a sensitive assay and mayprovide for enhanced detection, for example, of arrestin/GPCRs inendosomes. The assays using the modified GPCRs of the present inventionmay be useful for screening compounds and sample solutions for ligands,agonists, antagonists, inverse agonists, desensitization activecompounds, and the like. Once identified, these compounds may be usefulas drugs capable of modulating GPCR activity and useful in the treatmentof one or more of the disease states in which GPCRs have beenimplicated.

In a preferred assay according to the present invention, cells areprovided that express modified GPCRs of the present invention and thesecells may further contain a conjugate of an arrestin and a detectablemolecule.

Arrestin coupled to a detectable molecule may be detected and monitoredas it functions in the GPCR pathway. The location of the arrestin may bedetected, for example, evenly distributed in the cell cytoplasm,concentrated at a cell membrane, concentrated in clathrin-coated pits,localized on endosomes, and the like. In response to agoniststimulation, the proximity of arrestin to a GPCR may be monitored, aswell as the proximity to any other cell structure. For example, inresponse to agonist stimulation arrestin may be detected in proximity toGPCRs at a cell membrane, concentrated with GPCRs in clathrin-coatedpits, colocalized with a GPCR on endosomes, and the like.

The modified GPCRs of the present invention have an increased affinityfor arrestin and provide a stable complex of the GPCR with arrestin, andthereby promote colocalization of the GPCR with arrestin into endosomes.In the methods of assaying of the present invention, arrestin may bedetected, for example, in the cytoplasm, concentrated in proximity toGPCRs at a cell membrane, concentrated in proximity to GPCRs inclathrin-coated pits, colocalized with a GPCR on endosomes, and thelike. Preferably the arrestin may be detected colocalized with a GPCR onendosomes.

The association of arrestin with a GPCR at a cell membrane may berapidly detected after agonist addition, for example, approximately 1second to 2 minutes. The colocalization of arrestin with GPCR onendosomes may be detected within several minutes of agonist addition,for example, approximately 3 to 15 minutes, and may persist for extendedperiods of time, for example, after 1 hour. The association of arrestinwith GPCR on endosomes may give a strong, readily recognizable signal.Under magnification of 40× objective lens, the signal may bedoughnut-like in appearance. The signal resulting from thecompartmentalization of arrestin and GPCR colocalized in endosomesvesicles is typically easy to detect and may persist for extendedperiods of time.

A preferred method of assessing GPCR pathway activity of the presentinvention comprises (a) providing a cell that expresses at least onemodified GPCR of the present invention and that further comprises aconjugate of an arrestin and a detectable molecule; (b) inducingtranslocation of the arrestin; and (c) detecting interaction of thearrestin with the modified GPCR along the translocation pathway.

Interaction of the arrestin with the modified GPCR may be detected, forexample, in endosomes, in clathrin-coated pits, concentrated inproximity to a cell membrane, and the like. Preferably, interaction ofthe arrestin with the modified GPCR is detected in endosomes.Interaction of arrestin with a GPCR in endosomes may be detected withinseveral minutes of agonist addition, for example, approximately 3 to 15minutes, and may persist for extended periods of time, for example,after 1 hour. The association of arrestin with a GPCR in endosomes maygive a strong, readily recognizable signal that persists for extendedperiods of time.

In a method of screening compounds for GPCR activity of the presentinvention a cell that expresses at least one modified GPCR is provided.The cell further contains arrestin conjugated to a detectable molecule.The cell is exposed to the compounds to be tested. The location of thearrestin within the cell is detected. The location of the arrestinwithin the cell in the presence of the compound is compared to thelocation of the arrestin within the cell in the absence of the compound,and a difference is correlated between (1) the location of the arrestinwithin the cell in the presence of the compound and (2) the location ofthe arrestin within the cell in the absence of the compound.

By way of example, compounds and sample solutions may be screened forGPCR agonist activity using the modified GPCRs of the present invention.In this method, cells that express at least one modified GPCR of thepresent invention and that further comprise a conjugate of an arrestinand a detectable molecule are provided. The cells are exposed tocompounds or sample solutions to be tested. It is detected whetherinteraction of the arrestin with the modified GPCR is increased afterexposure to the test compound or solution, an increase in interactionbeing an indication that the compound or solution has GPCR agonistactivity. Interaction of the arrestin with the GPCR may be detected inendosomes, in clathrin-coated pits, in proximity to a cell membrane, andthe like. The modified GPCR may also be conjugated to a detectablemolecule, preferably at the carboxyl-terminus. As explained abovemodifications to GPCRs as in the present invention should not affect theGPCRs' natural affinity for agonists or ligands.

Also by way of example, compounds and sample solutions may be screenedfor GPCR antagonist or inverse agonist activity using the modified GPCRsof the present invention. Cells that express at least one modified GPCRof the present invention and that further comprise a conjugate of anarrestin and a detectable molecule are provided. The cells are exposedto compounds or sample solutions to be tested and to a known agonist forthe GPCR. It is detected whether interaction of the arrestin with themodified GPCR is decreased after exposure to the test compound orsolution, a decrease in interaction being an indication that thecompound or solution has GPCR antagonist or inverse agonist activity.Interaction of the arrestin with the GPCR may be detected in endosomes,in clathrin-coated pits, in proximity to a cell membrane, and the like.The modified GPCR may also be conjugated to a detectable molecule,preferably at the carboxyl-terminus. As explained above modifications toGPCRs as in the present invention should not affect the GPCRs' naturalaffinity for antagonists or inverse agonists.

Further by way of example, compounds and sample solutions may bescreened for GPCR desensitization activity using the modified GPCRs ofthe present invention. First cells that express at least one firstmodified GPCR of the present invention and that further comprise aconjugate of an arrestin and a detectable molecule are provided. Thefirst cells are exposed to compounds or sample solutions to be testedand to a known agonist for the first GPCR. It is detected whetherinteraction of the arrestin with the first modified GPCR is decreased ornot increased after exposure to the test compound or solution, adecrease or lack of increase in interaction being an indication that thecompound or solution has GPCR desensitization activity. Interaction ofthe arrestin with the GPCR may be detected in endosomes, inclathrin-coated pits, in proximity to a cell membrane, and the like.Then second cells that express at least one second modified GPCR of thepresent invention and that further comprise a conjugate of an arrestinand a detectable molecule are provided. The second modified GPCR is notrelated to the first modified GPCR. The second cells are exposed to thecompounds or sample solutions to be tested and to a known agonist forthe second GPCR. It is detected whether interaction of the arrestin withthe second modified GPCR is decreased or not increased after exposure tothe test compound or solution, a decrease or lack of increase ininteraction being an indication that the compound or solution has GPCRdesensitization activity independent of the GPCR expressed. Interactionof the arrestin with the GPCR may be detected in endosomes, inclathrin-coated pits, in proximity to a cell membrane, and the like.

The methods of assessing GPCR pathway activity of the present inventionalso include cell-free assays. In cell-free assays of the presentinvention, a substrate having deposited thereon a modified GPCR of thepresent invention is provided. A fluid containing a conjugate of anarrestin and a detectable molecule is also provided. Translocation ofthe arrestin is induced and interaction of the arrestin with the GPCR isdetected. The GPCR and arrestin may be obtained from whole cells andused in the cell-free assay after purification. The modified GPCR hasarrestin binding sites and agonist binding sites and may be supported ina multilayer or bilayer lipid vesicle. The vesicle supporting themodified GPCR may be deposited on the substrate, and the modified GPCRmay be supported in the lipid vesicle and deposited on the substratesuch that the arrestin binding sites are exposed to arrestin and thereceptor binding sites are accessible to agonists. The substrate may beany artificial substrate on which the GPCR may be deposited, includingbut not limited to, glass, plastic, diamond, ceramic, semiconductor,silica, fiber optic, diamond, biocompatible monomer, biocompatiblepolymer, polymer beads (including organic and inorganic polymers), andthe like.

The present invention relates to the compounds identified as ligands,agonists, antagonists, inverse agonists, or DACs by the methods ofassaying of the present invention. These compounds may be used to treatany one of the disease states in which GPCRs have been implicated. Thecompounds identified may be administered to a human or a non-human intherapeutically effective doses to treat or ameliorate a condition,disorder, or disease in which GPCRs have been implicated. Atherapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms of such a condition,disorder or disease.

Methods to Identify Compounds that Modulate GPCR Desensitization

The present invention relates to methods of screening for compounds thatmodulate GPCR desensitization. The methods utilize modified GRKs whichconstitutively phosphorylate GPCRs, resulting in constitutivedesensitization. These may be used to identify compounds that alter thedesensitization of GPCRs, even if the GPCR agonist is unknown. Onceidentified, these compounds may be useful as drugs capable of modulatingGPCR activity and useful in the treatment of one or more of the diseasestates in which GPCRs have been implicated.

In a preferred method according to the present invention, cells areprovided that contain an expression system and a nucleic acid encoding amodified GRK, resulting in constitutive desensitization of GPCRsexpressed in the cell. These cells may further contain an arrestinconjugated to a GFP.

A preferred method of identifying a compound which inhibits GPCRinternalization includes: (a) providing a cell including a GPCR, anarrestin, and a modified GRK; (b) exposing the cell to the compound(s);(c) determining the cellular distribution of the GPCR or arrestin; and(d) correlating a difference between (1) the location of the labeledmolecule in the cell in the presence of the compound(s) and (2) thelocation of the labeled molecule in the cell in the absence of thecompound(s) to modulation of GPCR internalization. Non-limitingembodiments of this method are described in FIGS. 4, 5, 6, and 7 andExamples 2, 3, 4, 5, 6, and 7.

The GRK of step (a), as described above, may be GRK 1, 2, 3, 4, 5, 6, orany other GRK, including splice variants, biologically active fragments,or modified GRKs. The GRK may be overexpressed and/or its expression maybe inducible. The GRK may include a CAAX motif.

In the above method, agonist may or may not be provided.

Methods of detecting the labeled molecules and determining the cellulardistribution of the GPCR or arrestin are described below.

GPCRs useful in the present invention include, but are not limited toGPCRs which have known agonists, GPCRs which do not have known agonists,GPCRs listed in FIG. 1A-1C, GPCRs illustrated in FIGS. 3, 4, 5, 6, and7, AT1AR, Class A GPCRs, Class B GPCRs, taste receptors, odorantreceptors, orphan receptors, modified GPCRs, GPCRs as described in U.S.patent application Ser. Nos. 10/054,616, 09/993,844, 10/095,620,10/101,235, 09/631,468, 10/141,725, 10/161,916, 09/469,554, 09/772,644,60/393,789, and 60/379,986, which are herein incorporated by reference,or biologically active fragments of the above GPCRs.

Vectors and Nucleic Acids, Host Cells for Protein Expression

The present invention relates to modified GRKs, including GRKs which areover-expressed, or their expression is inducible.

Nucleic acids encoding modified GRKs are provided. The present inventionrelates to the expression, over-expression, and the inducible expressionof these proteins. The expression may be carried out by a suitableexpression system contained in a vector, as described below.

One aspect of the present invention relates to the combination of (1)nucleic acids encoding a modified GRK with (2) a system for expressionof modified GRKs resulting in constitutive desensitization of GPCRs.This system for expression of modified GRKs may include a promoter ororigin of replication.

Another aspect of the present invention relates to modified GPCRs,nucleic acids encoding modified GPCRs, and host cell for modified GPCRexpression.

Nucleic acids encoding modified GPCRs are provided. The presentinvention relates to the expression, over-expression, and the inducibleexpression of these proteins. The expression may be carried out by asuitable expression system contained in a vector, as described below.

A feature of this invention is the expression of the DNA sequencesdisclosed herein. As is well known in the art, DNA sequences may beexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employing that expression vectorto transform an appropriate unicellular host. The transforming DNA mayor may not be integrated (covalently linked) into chromosomal DNA makingup the genome of the cell.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes, if not already part ofthe DNA sequence, the provision of an initiation codon, ATG, in thecorrect reading frame upstream of the DNA sequence.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences. Suitable vectors includederivatives of SV40 and known bacterial plasmids, e.g., E. coli plasmidscol El, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4;phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, andother phage DNA, e.g., M13 and filamentous single stranded phage DNA;yeast plasmids such as the 2μ plasmid or derivatives thereof, vectorsuseful in eukaryotic cells, such as vectors useful in insect ormammalian cells; vectors derived from combinations of plasmids and phageDNAs, such as plasmids that have been modified to employ phage DNA orother expression control sequences; and the like.

Any of a wide variety of expression control sequences—sequences thatcontrol the expression of a DNA sequence operatively linked to it—may beused in these vectors to express the DNA sequences of this invention.Such useful expression control sequences include, for example, the earlyor late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lacsystem, the trp system, the TAC system, the TRC system, the LTR system,the major operator and promoter regions of phage λ, the control regionsof fd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Strepromyces, fungi such as yeasts, plant cells,nematode cells, and animal cells, such as HEK-293, U2OS, CHO, R1.I, B-Wand L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7,BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells andplant cells in tissue culture. In one aspect of the present invention,the host cells include a GRK-C20 and an arrestin. In a further aspect ofthe present invention, the host cells include a GRK-C20, an arrestin,and a GPCR.

It will be understood that not all vectors, expression control sequencesand hosts will function equally well to express the DNA sequences ofthis invention. Neither will all hosts function equally well with thesame expression system. However, one skilled in the art will be able toselect the proper vectors, expression control sequences, and hostswithout undue experimentation to accomplish the desired expressionwithout departing from the scope of this invention. For example, inselecting a vector, the host must be considered because the vector mustfunction in it. The vector's copy number, the ability to control thatcopy number, and the expression of any other proteins encoded by thevector, such as antibiotic markers, will also be considered.

In selecting an expression control sequence, a variety of factors willnormally be considered. These include, for example, the relativestrength of the system, its controllability, and its compatibility withthe particular DNA sequence or gene to be expressed, particularly asregards potential secondary structures. Suitable unicellular hosts willbe selected by consideration of, e.g., their compatibility with thechosen vector, their secretion characteristics, their ability to foldproteins correctly, and their fermentation requirements, as well as thetoxicity to the host of the product encoded by the DNA sequences to beexpressed, and the ease of purification of the expression products.

Considering these and other factors a person skilled in the art will beable to construct a variety of vector/expression control sequence/hostcombinations that will express the DNA sequences of this invention onfermentation or in large scale animal culture.

It is further intended that modified GRK analogs may be prepared fromnucleotide sequences of the protein complex/subunit derived within thescope of the present invention. Analogs, such as fragments, may beproduced, for example, by pepsin digestion of GRK material. Otheranalogs, such as muteins, can be produced by standard site-directedmutagenesis of GRK coding sequences. Analogs exhibiting “GRK activity”such as small molecules, whether functioning as promoters or inhibitors,may be identified by known in vivo and/or in vitro assays.

As mentioned above, a DNA sequence encoding a modified GRK6 can beprepared synthetically rather than cloned. The DNA sequence can bedesigned with the appropriate codons for the GRK amino acid sequence. Ingeneral, one will select preferred codons for the intended host if thesequence will be used for expression. The complete sequence is assembledfrom overlapping oligonucleotides prepared by standard methods andassembled into a complete coding sequence. See, e.g., Edge, Nature,292:756 (1981); Nambair et al., Science, 223:1299 (1984); Jay et al., J.Biol. Chem., 259:6311 (1984).

Synthetic DNA sequences allow convenient construction of genes whichwill express GRK analogs or “muteins”. Alternatively, DNA encodingmuteins can be made by site-directed mutagenesis of native or modifiedGRK genes or cDNAs, and muteins can be made directly using conventionalpolypeptide synthesis.

A general method for site-specific incorporation of unnatural aminoacids into proteins is described in Christopher J. Noren, Spencer J.Anthony-Cahill, Michael C. Griffith, Peter G. Schultz, Science,244:182-188 (April 1989). This method may be used to create analogs withunnatural amino acids.

Additional motifs, such as epitope tags or sequences to aid inpurification, may be incorporated into the nucleic acids encoding themodified GRKs or modified GPCRs. Preferably, the nucleic acids encodingthe motifs may be at the 5′ or 3′ end of the nucleic acid, resulting inthe presence of the motif at the N or C terminus of the protein.

The Conjugates

The cells used in the methods of assaying of the present invention maycomprise a conjugate of an arrestin protein and a detectable molecule.In the cells and methods of the present invention, the cells may alsocomprise a conjugate of a modified GPCR of the present invention and adetectable molecule.

All forms of arrestin, naturally occurring and engineered variants,including but not limited to, visual arrestin, cone arrestin, βarrestin1 and βarrestin 2, may be used in the present invention. The modifiedGPCRs of the present invention may interact to a detectable level withall forms of arrestin.

Detectable molecules that may be used to conjugate with the arrestininclude, but are not limited to, molecules that are detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,radioactive, and optical means, including but not limited tobioluminescence, phosphorescence, and fluorescence. Detectable moleculesinclude, but are not limited to, GFP, luciferase, β-galactosidase,rhodamine-conjugated antibody, and the like. Detectable moleculesinclude radioisotopes, epitope tags, affinity labels, enzymes,fluorescent groups, chemiluminescent groups, and the like. Detectablemolecules include molecules which are directly or indirectly detected asa function of their interaction with other molecule(s). These detectablemolecules should be a biologically compatible molecule and should notcompromise the ability of the arrestin to interact with the GPCR systemand the interaction of the arrestin with the GPCR system must notcompromise the ability of the detectable molecule to be detected.Preferred detectable molecules are optically detectable molecules,including optically detectable proteins, such that they may be excitedchemically, mechanically, electrically, or radioactively to emitfluorescence, phosphorescence, or bioluminescence. More preferreddetectable molecules are inherently fluorescent molecules, such asfluorescent proteins, including, for example, Green Fluorescent Protein(GFP). The detectable molecule may be conjugated to the arrestin proteinby methods as described in Barak et al. (U.S. Pat. Nos. 5,891,646 and6,110,693). The detectable molecule may be conjugated to the arrestin atthe front-end, at the back-end, or in the middle.

The GPCR or biologically active fragments thereof may also be conjugatedwith a detectable molecule. Preferably, the carboxyl-terminus of theGPCR is conjugated with a detectable molecule. A carboxyl-terminal tailconjugated or attached to a detectable molecule can be used in acarboxyl-terminal tail exchange to provide the detectably labeled GPCR.

If the GPCR is conjugated with a detectable molecule, proximity of theGPCR with the arrestin may be readily detected. In addition, if the GPCRis conjugated with a detectable molecule, compartmentalization of theGPCR with the arrestin may be readily confirmed. The detectable moleculeused to conjugate with the GPCRs may include those as described above,including, for example, optically detectable molecules, such that theymay be excited chemically, mechanically, electrically, or radioactivelyto emit fluorescence, phosphorescence, or bioluminescence. Preferredoptically detectable molecules may be detected by immunofluorescence,luminescence, fluorescence, and phosphorescence.

For example, the GPCRs may be antibody labeled with an antibodyconjugated to an immunofluorescence molecule or the GPCRs may beconjugated with a luminescent donor. In particular, the GPCRs may beconjugated with, for example, luciferase, for example, Renillaluciferase, or a rhodamine-conjugated antibody, for example,rhodamine-conjugated anti-HA mouse monoclonal antibody. Preferably, thecarboxyl-terminal tail of the GPCR may be conjugated with a luminescentdonor, for example, luciferase. The GPCR, preferably thecarboxyl-terminal tail, also may be conjugated with GFP as described inL. S. Barak et al. “Internal Trafficking and Surface Mobility of aFunctionally Intact β₂-Adrenergic Receptor-Green Fluorescent ProteinConjugate”, Mol. Pharm. (1997) 51, 177-184.

Cell Types and Substrates

The cells of the present invention may express at least one modifiedGPCR of the present invention. The cells may further comprise aconjugate of an arrestin protein and a detectable molecule. Useful cellsinclude eukaryotic and prokaryotic cells, including, but not limited to,bacterial cells, yeast cells, fungal cells, insect cells, nematodecells, plant cells, and animal cells. Suitable animal cells include, butare not limited to, HEK-293 cells, U2OS cells, HeLa cells, COS cells,and various primary mammalian cells. An animal model expressing aconjugate of an arrestin and a detectable molecule throughout itstissues or within a particular organ or tissue type, may also be used.

The cells of the present invention may express one modified protein thatresults in agonist-independent localization of GPCRs to endocyticvesicles or endosomes.

A substrate may have deposited thereon a plurality of cells of thepresent invention. The substrate may be any suitable biologicallysubstrate, including but not limited to, glass, plastic, ceramic,semiconductor, silica, fiber optic, diamond, biocompatible monomer, orbiocompatible polymer materials.

Methods of Detection

Methods of detecting the intracellular location of the detectablylabeled arrestin, the intracellular location of a detectably labeledGPCR, or interaction of the detectably labeled arrestin, or other memberof GPCR/arrestin complex with a GPCR or any other cell structure,including for example, the concentration of arrestin or GPCR at a cellmembrane, colocalization of arrestin with GPCR in endosomes, andconcentration of arrestin or GPCR in clathrin-coated pits, and the like,will vary dependent upon the detectable molecule(s) used.

One skilled in the art readily will be able to devise detection methodssuitable for the detectable molecule(s) used. For optically detectablemolecules, any optical method may be used where a change in thefluorescence, bioluminescence, or phosphorescence may be measured due toa redistribution or reorientation of emitted light. Such methodsinclude, for example, polarization microscopy, BRET, FRET, evanescentwave excitation microscopy, and standard or confocal microscopy.

In a preferred embodiment arrestin may be conjugated to GFP and thearrestin-GFP conjugate may be detected by confocal microscopy. Inanother preferred embodiment, arrestin may conjugated to a GFP and theGPCR may be conjugated to an immunofluorescent molecule, and theconjugates may be detected by confocal microscopy. In an additionalpreferred embodiment, arrestin may conjugated to a GFP and thecarboxy-terminus of the GPCR may be conjugated to a luciferase and theconjugates may be detected by bioluminescence resonance emissiontechnology. In a further preferred embodiment arrestin may be conjugatedto a luciferase and GPCR may be conjugated to a GFP, and the conjugatesmay be detected by bioluminescence resonance emission technology. Themethods of the present invention are directed to detecting GPCRactivity. The methods of the present invention allow enhanced monitoringof the GPCR pathway in real time.

In a preferred embodiment, the localization pattern of the detectablemolecule is determined. In a further preferred embodiment, alterationsof the localization pattern of the detectable molecule may bedetermined. The localization pattern may indicate cellular distributionof the detectable molecule. Certain methods of detection are describedin U.S. Ser. No. 10/095,620, filed Mar. 12, 2002, which claims priorityto U.S. Provisional Patent Application No. 60/275,339, filed Mar. 13,2001, the contents of which are incorporated by reference in theirentirety.

Molecules may also be detected by their interaction with anotherdetectably labeled molecule, such as an antibody.

Disease Treatment

Another aspect of the invention relates to methods of treating a humanor non-human subject suffering from a GPCR-related disease, such ascardiovascular disease, heart failure, asthma, nephrogenic diabetesinsipidus, or hypertension. For example, compounds which alter AT1ARinternalization may be useful to treat diseases and conditions relatedto AT1AR. Such diseases and conditions related to AT1AR include, but arenot limited to: renal disease, diabetes and nephropathy, diabetesmellitus, type 2 diabetes, nephropathy, hypertension, congestive heartfailure, endothelial dysfunction, vascular inflammation, and variousheart diseases. Such treatment can be performed either by administeringto a subject in need of such treatment, an amount of the compoundsidentified by the present method sufficient to treat the GPCR-relateddisease, or at least to lessen the symptoms thereof.

Treatment may also be effected by administering to the subject the nakedmodified nucleic acid sequences of the invention, such as by directinjection, microprojectile bombardment, delivery via liposomes or othervesicles, or by means of a vector which can be administered by one ofthe foregoing methods. Gene delivery in this manner may be consideredgene therapy.

Pharmaceutical Compositions

The preparation of therapeutic compositions which contain polypeptides,analogs or active fragments as active ingredients is well understood inthe art. Typically, such compositions are prepared as injectables,either as liquid solutions or suspensions, however, solid forms suitablefor solution in, or suspension in, liquid prior to injection can also beprepared. The preparation can also be emulsified. The active therapeuticingredient is often mixed with excipients which are pharmaceuticallyacceptable and compatible with the active ingredient. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof. In addition, if desired, thecomposition can contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents which enhance theeffectiveness of the active ingredient.

Losartan is a known angiotensin receptor antagonist, of the followingformula:

The present invention describes a new use of losartan: losartan isuseful in methods of altering arrestin translocation to the AT1ARreceptor (FIGS. 8-9). This method my be used to treat a patient byadministering an effective amount of losartan to a patient in needthereof.

A GPCR agonist, antagonist, or DAC obtained by the methods disclosedherein can be formulated into the therapeutic composition as neutralizedpharmaceutically acceptable salt forms. Pharmaceutically acceptablesalts include the acid addition salts (formed with the free amino groupsof the polypeptide or antibody) and which are formed with inorganicacids such as, for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric, mandelic, and the like. Saltsformed from the free carboxyl groups can also be derived from inorganicbases such as, for example, sodium, potassium, ammonium, calcium, orferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The therapeutic compositions are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for humans, each unit containing a predetermined quantityof active material calculated to produce the desired therapeutic effectin association with the required diluent (i.e., carrier, or vehicle).

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compositions lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compositionused in the method of the invention, the therapeutically effective dosecan be estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range which includes the IC50 (i.e., the concentration ofthe test composition which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered depends on the subject to be treated, capacity of thesubject's immune system to utilize the active ingredient, and degree ofmodulation of GPCR activity desired. Precise amounts of activeingredient required to be administered depend on the judgment of thepractitioner and are peculiar to each individual. However, suitabledosages may range from about 0.001 to 30, preferably about 0.01 to about25, and more preferably about 0.1 to 20 milligrams of active ingredientper kilogram body weight of individual per day and depend on the routeof administration. Suitable regimes for initial administration andbooster shots are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain concentrations often nanomolar to ten micromolar in the blood are contemplated.

The skilled artisan will appreciate that certain factors may influencethe dosage required to effectively treat a subject, including but notlimited to, the severity of the disease or condition, disorder, ordisease, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of the composition(s) caninclude a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with thecomposition in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of the composition used for treatment may increaseor decrease over the course of a particular treatment. Changes in dosagemay result and become apparent from the results of diagnostic assays asdescribed herein.

The therapeutic compositions may further include an effective amount ofthe GPCR agonist, antagonist, or DAC and one or more of the followingactive ingredients: an antibiotic, a steroid, and the like.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention can be prepared as SATE((S-acetyl-2-thioethyl) phosphate) derivatives according to the methodsdisclosed for example in WO 93/24510 and in WO 94/26764.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Thecompounds for modulating any of the disclosed genes, gene transcripts orproteins encoded thereby include antisense compounds as well as othermodulatory compounds.

Pharmaceutically acceptable base addition salts for use with antisenseas well as other modulatory compounds are formed with metals or amines,such as alkali and alkaline earth metals or organic amines. Examples ofmetals used as cations are sodium, potassium, magnesium, calcium, andthe like. Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, dicyclohexylamine,ethylenediamine, N-methylglucamine, and procaine (see, e.g., Berge etal., “Pharmaceutical Salts,” J. Pharma. Sci., 1977, 66: 1-19). The baseaddition salts of acidic compounds are prepared by contacting the freeacid form with a sufficient amount of the desired base to produce thesalt in the conventional manner. The free acid form may be regeneratedby contacting the salt form with an acid and isolating the free acid inthe conventional manner. The free acid forms differ from theirrespective salt forms somewhat in certain physical properties such assolubility in polar solvents, but otherwise the salts are equivalent totheir respective free acid for purposes of the present invention. Asused herein, a pharmaceutical addition salt includes a pharmaceuticallyacceptable salt of an acid form of one of the components of thecompositions of the invention. These include organic or inorganic acidsalts of the amines. Preferred acid salts are the hydrochlorides,acetates, salicylates, nitrates and phosphates. Other suitablepharmaceutically acceptable salts are known in the art and include basicsalts of a variety of inorganic and organic acids, such as, for example,with inorganic acids (e.g., hydrochloric acid, hydrobromic acid,sulfuric acid or phosphoric acid); with organic carboxylic, sulfonic,sulfo- or phospho-acids or N-substituted sulfamic acids, for exampleacetic acid, propionic acid, glycolic acid, succinic acid, maleic acid,hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid,tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid,glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid,2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinicacid; and with amino acids, such as the 20 alpha-amino acids involved inthe synthesis of proteins in nature, for example glutamic acid oraspartic acid, and also with phenylacetic acid, methanesulfonic acid,ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonicacid, benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid.

Pharmaceutically acceptable salts of compounds may also be prepared witha pharmaceutically acceptable cation. Suitable pharmaceuticallyacceptable cations are well known in the art and include alkaline,alkaline earth, ammonium and quaternary ammonium cations. Carbonates orhydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

The antisense compounds and other modulatory compounds described hereincan be utilized in pharmaceutical compositions by adding an effectiveamount of an antisense compound or other modulatory compound to asuitable pharmaceutically acceptable diluent or carrier. Use of thecompounds and methods of the invention may also be usefulprophylactically.

The antisense compounds of the invention are useful for research anddiagnostics, because these compounds hybridize to nucleic acids encodinga gene identified using the systematic discovery technique or a mRNAtranscript thereof. Such hybridization allows the use of sandwich andother assays to easily be constructed to exploit this fact.Hybridization of the antisense oligonucleotides of the invention with anucleic acid encoding a gene or gene transcript identified by asystematic discovery method can be detected by means known in the art.Such means may include, for example, conjugation of an enzyme to theoligonucleotide, radiolabelling of the oligonucleotide or any othersuitable detection means. Kits using such detection means for detectingthe level of a transcript of a gene in a sample may also be prepared.

The present invention also includes pharmaceutical antisensecompositions and formulations which include the antisense compounds andother modulatory compounds and compositions of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated.

In certain embodiments, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment. This may be achieved by, for example, and not by way oflimitation, local infusion during surgery, topical application, e.g., inconjunction with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. In oneembodiment, administration can be by direct injection at the site (orformer site) of a malignant tumor or neoplastic or pre-neoplastictissue.

For topical application, the compositions may be combined with a carrierso that an effective dosage is delivered, based on the desired activity.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer, salts,flavoring, coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active composition.

The compositions may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

For administration by inhalation, the compositions for use according tothe present invention are conveniently delivered in the form of anaerosol spray, presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compositionand a suitable powder base such as lactose or starch.

The compositions may also be formulated as a depot preparation. Suchlong acting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the compositions may be formulated withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

The compositions may, if desired, be presented in a pack or dispenserdevice that may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

Pharmaceutical compositions (e.g., gene, gene transcript or proteinproduct modulatory agents as described herein) of the present inventioninclude, but are not limited to, solutions, emulsions, andliposome-containing formulations. These compositions may be generatedfrom a variety of components that include, but are not limited to,preformed liquids, self-emulsifying solids and self-emulsifyingsemisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

In one embodiment of the present invention, the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature, these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1 m indiameter. See, e.g., Idson, in Pharmaceutical Dosage Forms v. 1, p. 199(Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork); Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245; Block inPharmaceutical Dosage Forms, v. 2, p. 335; Higuchi et al., inRemington's Pharmaceutical Sciences 301 (Mack Publishing Co., Easton,Pa., 1985). Emulsions are often biphasic systems comprising of twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions may be either water-in-oil (w/o) or of theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and antioxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms v. 1, p. 199 (Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms, v.1, p. 285; Idson, in Pharmaceutical Dosage Forms, v. 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants may beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (Rieger, inPharmaceutical Dosage Forms).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers, especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, non-swelling clays (e.g.,bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate), pigmentsand nonpolar solids (e.g., carbon or glyceryl tristearate).

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, v. 1 p. 385 (Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York)).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers, such as polysaccharides (e.g., acacia, agar,alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (e.g., carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (e.g., carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers (e.g., tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene) or reducing agents (e.g.,ascorbic acid and sodium metabisulfite), and antioxidant synergists(e.g., citric acid, tartaric acid, and lecithin).

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, v. 1, p. 199).Emulsion formulations for oral delivery have been very widely usedbecause of reasons of ease of formulation, efficacy from an absorptionand bioavailability standpoint. (Rosoff, in Pharmaceutical Dosage Forms,v. 1, p. 245 (Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York); Idson, in Pharmaceutical Dosage Forms). Mineral-oilbase laxatives, oil-soluble vitamins and high fat nutritive preparationsare among the materials that have commonly been administered orally aso/w emulsions.

In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, v. 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in Controlled Release of Drugs; Polymers and Aggregate Systems,185-215 (Rosoff, M., Ed., 1989, VCH Publishers, New York).Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, 271 (Mack Publishing Co., Easton,Pa., 1985).

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with co-surfactants. The co-surfactant, usuallya short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules.

Microemulsions may, however, be prepared without the use ofco-surfactants and alcohol-free self-emulsifying microemulsion systemsare known in the art. The aqueous phase may typically be, but is notlimited to, water, an aqueous solution of the drug, glycerol, PEG300,PEG400, polyglycerols, propylene glycols, and derivatives of ethyleneglycol. The oil phase may include, but is not limited to, materials suchas Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain(C8-C12) mono-, di-, and tri-glycerides, polyoxyethylated glyceryl fattyacid esters, fatty alcohols, polyglycolized glycerides, saturatedpolyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharm. Res., 1994, 11:1385-90; Ritschel, Meth. Find. Exp. Clin.Pharmacol., 1993, 13: 205). Microemulsions afford advantages of improveddrug solubilization, protection of drug from enzymatic hydrolysis,possible enhancement of drug absorption due to surfactant-inducedalterations in membrane fluidity and permeability, ease of preparation,ease of oral administration over solid dosage forms, improved clinicalpotency, and decreased toxicity (Constantinides et al., 1994; Ho et al.,J. Pharm. Sci., 1996, 85: 138-143). Often microemulsions may formspontaneously when their components are brought together at ambienttemperature. This may be particularly advantageous when formulatingthermolabile drugs, peptides or oligonucleotides. Microemulsions havealso been effective in the transdermal delivery of active components inboth cosmetic and pharmaceutical applications. It is expected that themicroemulsion compositions and formulations of the present inventionwill facilitate the increased systemic absorption of oligonucleotidesand nucleic acids and other active agents from the gastrointestinaltract, as well as improve the local cellular uptake of oligonucleotidesand nucleic acids and other active agents within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Crit. Rev. Therap. Drug Carrier Systems, 1991, p. 92). Each of theseclasses has been discussed above.

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, are useful because of their specificity and the duration ofaction. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo. Selection of theappropriate liposome depending on the agent to be encapsulated would beevident given what is known in the art.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include: (a) liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; (b) liposomescan incorporate a wide range of water and lipid soluble drugs; (c)liposomes can protect encapsulated drugs in their internal compartmentsfrom metabolism and degradation (Rosoff, in Pharmaceutical DosageForms). Important considerations in the preparation of liposomeformulations are the lipid surface charge, vesicle size and the aqueousvolume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes. As the mergingof the liposome and cell progresses, the liposomal contents are emptiedinto the cell where the active agent may act.

Another embodiment also contemplates the use of liposomes for topicaladministration. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin. Several reports have detailed the ability ofliposomes to deliver agents including high-molecular weight DNA into theskin. Compounds including analgesics, antibodies, hormones andhigh-molecular weight DNAs have been administered to the skin. Themajority of applications resulted in the targeting of the upperepidermis.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Comm., 1987, 147:980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al., J.Controlled Release, 1992, 19: 269-74).

Another contemplated liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

“Sterically stabilized” liposomes, which refers to liposomes comprisingone or more specialized lipids that, when incorporated into liposomes,result in enhanced circulation lifetimes relative to liposomes lackingsuch specialized lipids are also contemplated. Examples of stericallystabilized liposomes are those in which part of the vesicle-forminglipid portion of the liposome (A) comprises one or more glycolipids,such as monosialoganglioside GM1, or (B) is derivatized with one or morehydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Whilenot wishing to be bound by any particular theory, it is thought in theart that, at least for sterically stabilized liposomes containinggangliosides, sphingomyelin, or PEG-derivatized lipids, the enhancedcirculation half-life of these sterically stabilized liposomes derivesfrom a reduced uptake into cells of the reticuloendothelial system (RES)(Allen et al., FEBS Lett., 1987, 223: 42; Wu et al., Can. Res., 1993,53: 3765).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. See, e.g., Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53:2778) described liposomes comprising a nonionic detergent, 2C12 15G,that contains a PEG moiety. Ilium et al. (FEBS Lett., 1984, 167: 79)noted that hydrophilic coating of polystyrene particles with polymericglycols results in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268: 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029: 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 BIand WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by, e.g., Woodle et al. (U.S. Pat. Nos. 5,013,556 and5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and EuropeanPatent No. EP 0 496 813 B1). Liposomes comprising a number of otherlipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No.5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky et al.).Liposomes comprising PEG-modified ceramide lipids are described in WO96/10391 (Choi et al.). U.S. Pat. No. 5,540,935 (Miyazaki et al.) andU.S. Pat. No. 5,556,948 (Tagawa et al.) describe PEG-containingliposomes that can be further derivatized with functional moieties ontheir surfaces.

Methods of encapsulating nucleic acids in liposomes is also known in theart. See, WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, p. 285 (Marcel Dekker, Inc., New York,N.Y., 1988, p. 285)).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, 285 (MarcelDekker, Inc., New York, N.Y., 1988).

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids and otheragents, particularly oligonucleotides, to the skin of animals. Mostdrugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugsmay cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Another embodiment of the invention contemplates pharmaceuticalcompositions comprising surfactants. Surfactants (or “surface-activeagents”) are chemical entities which, when dissolved in an aqueoussolution, reduce the surface tension of the solution or the interfacialtension between the aqueous solution and another liquid, with the resultthat absorption of oligonucleotides through the mucosa is enhanced. Inaddition to bile salts and fatty acids, these penetration enhancersinclude, for example, sodium lauryl sulfate, polyoxyethylene-9-laurylether and polyoxyethylene-20-cetyl ether) (Lee et al., Crit. Rev.Therap. Drug Carrier Systems, 1991, 92); and perfluorochemicalemulsions, such as FC-43 (Takahashi et al., J. Pharm. Pharmacol., 1988,40: 252).

Another embodiment contemplates the use of various fatty acids and theirderivatives to act as penetration enhancers include, for example, oleicacid, lauric acid, capric acid (n-decanoic acid), myristic acid,palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate,tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylicacid, arachidonic acid, glycerol 1-monocaprate,1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-10 alkylesters thereof (e.g., methyl, isopropyl and t-butyl), and mono- anddi-glycerides thereof (i.e., oleate, laurate, caprate, myristate,palmitate, stearate, linoleate, and the like) (Lee et al., 1991;Muranishi, Crit. Rev. Therap. Drug Carrier Systems, 1990, 7: 1-33; ElHariri et al., J. Pharm. Pharmacol., 1992, 44: 651-4).

The compositions comprising the active agents of the invention mayfurther comprise bile salts. The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, N.Y.,1996, pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus, the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. The bile salts of the inventioninclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., 1991; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences,18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, 1990; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263: 25; Yamashita et al., J. Pharm. Sci., 1990, 79: 579-83).

The invention further contemplates compositions comprising chelatingagents. Chelating agents can be defined as compounds that removemetallic ions from solution by forming complexes therewith, with theresult that absorption of oligonucleotides through the mucosa isenhanced. With regards to their use as penetration enhancers for usewhen the active agent is an antisense agent, chelating agents have theadded advantage of also serving as DNase inhibitors, as mostcharacterized DNA nucleases require a divalent metal ion for catalysisand are thus inhibited by chelating agents (Jarrett, J. Chromatogr.,1993, 618: 315-39). Chelating agents of the invention include but arenot limited to disodium ethylenediaminetetraacetate (EDTA), citric acid,salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines) (Lee et al., 1991;Muranishi, 1990; Buur et al., J. Control Rel., 1990, 14: 43-51).

The invention also contemplates pharmaceutical compositions comprisingactive agents and non-chelating non-surfactants. Non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants, but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, 1990). Thisclass of penetration enhancers include, for example, unsaturated cyclicureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,1991); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39: 621-6).

For pharmaceutical compositions comprising oligonucleotides, agents thatenhance uptake of oligonucleotides at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal., U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes(e.g., limonene and menthone).

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate oligonucleotide in hepatic tissue can be reduced whenit is coadministered with polyinosinic acid, dextran sulfate,polycytidic acid or 4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonicacid (Miyao et al., Antisense Res. Dev., 1995, 5: 115-121; Takakura etal., Antisense & Nucl. Acid Drug Dev., 1996, 6: 177-183).

The pharmaceutical compositions disclosed herein may also comprise anexcipients. In contrast to carrier compounds described above, theseexcipients include a pharmaceutically acceptable solvent, suspendingagent or any other pharmacologically inert vehicle for delivering one ormore nucleic acids or other active agents to an animal. The excipientmay be liquid or solid and is selected, with the planned manner ofadministration in mind, so as to provide for the desired bulk,consistency, etc., when combined with a nucleic acid or other activeagent and the other components of a given pharmaceutical composition.Typical pharmaceutical carriers include, but are not limited to, bindingagents (e.g., pregelatinized maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and othersugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate,ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.);lubricants (e.g., magnesium stearate, talc, silica, colloidal silicondioxide, stearic acid, metallic stearates, hydrogenated vegetable oils,corn starch, polyethylene glycols, sodium benzoate, sodium acetate,etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); andwetting agents (e.g., sodium lauryl sulphate, etc.).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids and othercontemplated active agents may include sterile and non-sterile aqueoussolutions, non-aqueous solutions in common solvents such as alcohols, orsolutions of the nucleic acids in liquid or solid oil bases. Thesolutions may also contain buffers, diluents and other suitableadditives. Pharmaceutically acceptable organic or inorganic excipientssuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids or other contemplated active agents can beused.

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, e.g., antipruritics, astringents, local anestheticsor anti-inflammatory agents, or may contain additional materials usefulin physically formulating various dosage forms of the compositions ofthe present invention, such as dyes, flavoring agents, preservatives,antioxidants, opacifiers, thickening agents and stabilizers. However,such materials, when added, should not unduly interfere with thebiological activities of the components of the compositions of thepresent invention. The formulations can be sterilized and, if desired,mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, colorings, flavorings and/or aromatic substances andthe like which do not deleteriously interact with the nucleic acid(s) ofthe formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Test Kits

In a further embodiment of this invention, commercial test kitsincluding an assay system for screening potential drugs effective tomodulate the activity of the GPCR may be prepared. The test kits mayinclude cells, nucleic acids, or proteins described herein. The testkits may be used to carry out any of the methods described herein. AGPCR of interest may be introduced into host cells of the test kit. Thetest kit may be useful for determining if the GPCR is expressed at theplasma membrane, if the phosphorylated or unphosphorylated GPCR bindsarrestin, or if the phosphorylated or urphosphorylated GPCR isinternalized. The test kit may be useful for the identification ofcompounds that alter the desensitization of the GPCR of interest.

The GPCR may be introduced into a test system, and the prospective drugmay also be introduced into the resulting cell culture, and the culturethereafter examined to observe any changes in the GPCR activity (e.g.,signaling, recycling, affinity for arrestin, and the like) in the cells.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Experimental Procedures

The present inventors subcloned the Bovine GRK2-C20 cDNA (Inglese etal., Nature 1992) into the expression vector pcDNA3.lzeo+ (Invitrogen).Expression of this cDNA produces GRK2 with a CAAX motif (where C iscysteine, A is a small aliphatic residue, and X is an uncharged aminoacid) added to the carboxyl terminus. The specific CAAX motif added tothe end of GRK2, CVLL, directs the geranylgeranylation (C20isoprenylation) of this protein. The enzyme-directed covalent attachmentof the 20 carbon geranylgeranyl lipid group to the carboxyl terminus ofGRK2 allows it to be localized at the plasma membrane (Inglese et al.,Nature 1992).

Cell Culture

Human embryonic kidney (HEK-293) cells were purchased from the AmericanType Culture Collection (ATCC) and grown in Eagle's minimum essentialmedium (EMEM) supplemented with 10% (v/v) heat-inactivated fetal calfserum and gentamicin (100 μg/ml). HEK-293 cells stably expressingarrestin-GFP (HEK293-ArrGFP) were generated by standard procedures usingG418 selection (0.4 mg/ml). HEK-293 cells were transiently transfectedwith arrestin-GFP, the GPCR of interest, and GRK2-C20. For controlexperiments performed in parallel, HEK-293 cells were transientlytransfected with arrestin-GFP, the GPCR of interest, and no GRK2-C20.HEK293 cells were transiently transfected with arrestin-GFP, the GPCR ofinterest and GRK2-C20. For control experiments performed in parallel,the HEK293 cells were transiently transfected with arrestin-GFP weretransiently transfected with the GPCR of interest and no GRK2-C20. Alltransfections were performed by the calcium phosphate coprecipitationmethod as previously described (Oakley et al., 1999). Following thetransfection, cells were maintained in the culture medium (EMEMsupplemented with 10% FCS and 10 μg/ml gentamycin) for approximately 24hours. The cells were then plated on 35 mm glass bottom dishes (MatTek)and incubated for an additional 16-24 hours. Transfected GPCRs includedboth known GPCRs (receptors for which the natural ligand is know) andorphan GPCRs (receptors for which the natural ligand has not yet beenidentified).

Confocal Microscopy

Transfected HEK-293 cells were plated on 35 mm glass bottom dishes(MatTek) and cultured overnight. The next day, the medium was removedand replaced with serum-free medium supplemented with 10 mM HEPES for anadditional 1 hour incubation at 37° C. The distribution of arrestin-GFPwas then assessed using a Zeiss laser scanning confocal microscope (LSM5 Pascal). Images were acquired with a 63× oil objective from live cellsusing single line excitation (488 nm) and a LP505 emission filter.

Example 2 Agonist-Independent Desensitization of Known GPCRs UponExpression of a Modified GRK

The present inventors determined that overexpression of the GRK2-C20,which is expressed at the plasma membrane (Inglese et al., Nature 1992),in a cell line expressing arrestin-GFP promoted the binding ofarrestin-GFP to GPCRs in the absence of added ligand.

The HEK293 cells transiently transfected with arrestin-GFP weretransiently transfected with the GPCR of interest and with or withoutGRK2-C20. Using confocal microscopy, the distribution of thearrestin-GFP was determined. The localization of the arrestin-GFP atclathrin coated pits, endocytic vesicles, endosomes, or other stages inthe desensitization pathway indicated arrestin-GFP binding to the GPCR.Thus, GPCR desensitization, visualized by the binding of arrestin-GFP tothe GPCRs, was analyzed.

In the absence of added agonist, arrestin-GFP localized in small puncta(presumably clathrin coated pits) at the plasma membrane in cellsexpressing GRK2-C20 and either the cannabinoid type 2 receptor (CB2R)(FIG. 4). Moreover, in the absence of added agonist, arrestin-GFPlocalized in endocytic vesicles in cells expressing GRK2-C20 and eitherthe angiotensin II type IA receptor (AT1AR), vasopressin V2 receptor(V2R), (FIG. 4) or neurokinin-1/substance P receptor (NK-1 or SPR). Incontrol cells expressing each of the receptors (CB2R, AT1AR, V2R, orSPR) but lacking GRK2-C20, arrestin-GFP was diffusely expressed in thecytoplasm and did not localize to any significant extent in pits at theplasma membrane or vesicles inside the cell (FIG. 4).

Example 3 Agonist-Independent Desensitization of Orphan GPCRs UponExpression of a Modified GRK

The present inventors determined that overexpression of the GRK2-C20,which is expressed at the plasma membrane (Inglese et al., Nature 1992),in a cell line expressing arrestin-GFP promoted the binding ofarrestin-GFP to GPCRs in the absence of added ligand.

As above, the HEK293 cells transiently transfected with arrestin-GFPwere transiently transfected with the GPCR of interest and with orwithout GRK2-C20. Using confocal microscopy, the distribution of thearrestin-GFP was determined. The localization of the arrestin-GFP atclathrin coated pits, endocytic vesicles, endosomes, or other stages inthe desensitization pathway indicated arrestin-GFP binding to the GPCR.Thus, GPCR desensitization, visualized by the binding of arrestin-GFP tothe GPCRs, was analyzed.

In the absence of added agonist, arrestin-GFP localized in small puncta(presumably clathrin coated pits) at the plasma membrane in cellsexpressing the orphan receptor GPR55 (FIG. 7). In control cellsexpressing GPR55 but lacking GRK2-C20, arrestin-GFP was diffuselyexpressed in the cytoplasm and did not localize to any significantextent in pits at the plasma membrane or vesicles inside the cell (FIG.7). Other orphan GPCRs are described below.

Example 4 Method of Analyzing the Ability of a GPCR to Bind Arrestin

The present inventors developed a method to determine if a GPCR ofinterest is expressed at the plasma membrane. Preferably, the expressionof orphan GPCRs may be analyzed in host cells in which GPCRs desensitizein an agonist-independent manner, as described herein.

As above, the HEK293 cells transiently transfected with arrestin-GFPwere transiently transfected with the GPCR of interest and with orwithout GRK2-C20. Using confocal microscopy, the distribution of thearrestin-GFP was determined. The localization of the arrestin-GFP atclathrin coated pits, endocytic vesicles, endosomes, or other stages inthe desensitization pathway indicated arrestin-GFP binding to the GPCR.Thus, GPCR desensitization, visualized by the binding of arrestin-GFP tothe GPCRs, was analyzed.

Certain GPCRs, as described above, localized in clathrin coated pits,endocytic vesicles, endosomes, or other stages in the desensitizationpathway. This localization indicated that the GPCRs had the ability tobind arrestin, because arrestin binding is requisite for subsequentlocalization in the desensitization pathway. A GPCR that does not bindarrestin would not enter or localize in the desensitization pathway.GPCRs that do not bind arrestin may be altered such that they do bindarrestin. The present inventors modified certain GPCRs to enhancearrestin affinity, as described below.

Example 5 Method of Increasing the Ability of a GPCR to Bind Arrestin

The present inventors modified GPCRs to enhance their binding toarrestin. These modifications are described in U.S. Ser. No. 09/993,844.GPCRs were modified at their C-terminal tails to be betterphosphorylated by GRKs. These modified and phosphorylated GPCRs then hadenhanced binding to arrestin. They demonstrated increasedinternalization. The letter E (for enhanced phosphorylation) is added tothe end of the name of the GPCR which has been modified in this manner.

Modified GPCR constructs were generated by polymerase chain reactionfollowing standard protocols and contain the HA epitope. Chimericreceptors were constructed in which the carboxyl-terminal tails of theGPCR and V2R were exchanged (FIG. 3A-3BB), one for the other, after theputative sites of palmitoylation. Sequences of the DNA constructs wereconfirmed by DNA sequencing.

The nucleic acids of the GPCR of interest were PCR-amplified withprimers that introduced a Not I restriction enzyme site (gcggccgc)immediately after the codon for a cysteine residue (a putative site ofpalmitoylation) 10 to 25 amino acids (preferably 15 to 20) downstream ofthe NPXXY that is to be fused to the V2R carboxyl terminus. Theamplified receptor DNA fragment was then subcloned into the pEArrB-1vector (described in U.S. patent application Ser. No. 09/993,844) usingthe Not I restriction enzyme site and an additional restriction enzymesite upstream of the receptor atg start codon. When expressed, themodified GPCR will contain a 31 amino acid peptide fused to the receptorcarboxyl terminus. The first two amino acids will be Ala residuescontributed by the Not I site, and the last 29 amino acids will be fromthe V2R carboxyl terminus.

The present inventors modified the carboxyl-terminal tails of thefollowing receptors as described above and in the U.S. patentapplication Ser. No. 09/993,844: the β2-adrenergic receptor (β2ARE),dopamine D1A receptor (D1ARE), mu opiod receptor (MORE), orphan GPR3(GPR3E), orphan GPR6 (GPR6E), orphan GPR12 (GPR12E), orphan GPR7(GPR7E), orphan GPR8 (GPR8E), orphan GPR55 (GPR55E), orphan SREB2(SREB2E), and orphan SREB3 (SREB3E). The “E” stands for “enhancedarrestin binding”. In the absence of added agonist, arrestin-GFPlocalized in endocytic vesicles for each of the modified GPCRs listedabove when co-expressed with GRK2-C20 (FIGS. 4, 5, 6, and 7). For someof these modified receptors (such as orphan GPR6E), a small butsignificant amount of arrestin-GFP was observed to localize inintracellular vesicles in the control cells lacking the GRK2-C20 (FIG.5). However, overexpression of GRK2-C20 with these receptors promoted amarked increase in this response (FIG. 5). The amino acid and nucleicacid sequences of these modified GPCRs, the wild-type sequences, andsequences of HA-tagged modified GPCRs are shown in FIG. 3A-3BB and inSEQ ID Nos: 35-90.

Example 6 Method of Determining if a GPCR of Interest is Expressed atthe Plasma Membrane

The present inventors developed a method to determine if a GPCR ofinterest is expressed at the plasma membrane.

The HEK293 cells transiently transfected with arrestin-GFP weretransiently transfected with the GPCR of interest and with or withoutGRK2-C20. Using confocal microscopy, the distribution of thearrestin-GFP was determined. The localization of the arrestin-GFP atclathrin coated pits, endocytic vesicles, endosomes, or other stages inthe desensitization pathway indicated arrestin-GFP binding to the GPCR.Thus, GPCR desensitization, visualized by the binding of arrestin-GFP tothe GPCRs, was analyzed.

Certain GPCRs, as described above, localized in clathrin coated pits,endocytic vesicles, endosomes, or other stages in the desensitizationpathway. This localization indicated that the GPCRs were expressed atthe plasma membrane, because plasma membrane expression is requisite forsubsequent localization in the desensitization pathway. A GPCR that wasnot expressed at the plasma membrane would not localize in thedesensitization pathway. GPCRs that do not express at the plasmamembrane may be altered such that they do express at the plasmamembrane. For example, the expression of the GPCR may be altered, theamino acid sequence of the GPCR may be altered, or the GPCR may beintroduced into another host cell.

Example 7 Monitoring Desensitization of GPCR Mutants

Desensitization may be monitored in cells including GPCR mutants. Thedesensitization of the GPCR mutant may be dependent on GRKoverexpression.

A vector including the human β₂AR-E-Y326A containing a point mutation,the Tyrosine residue 326 converted to Alanine, will be transfected intocells expressing arrestin-GFP and a GRK, which may be modified. The “E”indicates that the GPCR has been modified, as described above. The Y326Amutation causes the GPCR to be dependent on overexpressed GRK forphosphorylation and subsequent desensitization. The β₂AR-Y326A willdesensitize in the absence of agonist upon expression of GRK-C20. Theexpression of the GRK may be altered, including methods of altering theamount of GRK nucleic acids in the cell using an inducible promoter,replication controlling machinery such as the origin of replication, ormanually altering the amount of vector in the cells.

The cells will be seeded in 96 well or higher density plates andincubated overnight. The next morning the activator of the induciblesystem or vehicle only will be added to the wells to induceoverexpression of the GRK or modified GRK. Agonist will be added tocells expressing the GRK (not the modified GRK).

Compounds of interest will then be added to the wells to see if theyalter the internalization of arrestin-GFP. The cells will then be fixedwith 2% paraformaldehyde and the amount of arrestin-GFP translocationwill be measured using image analysis systems.

Example 8 Non Peptide Antagonist/Inverse GPCR Agonist InhibitsConstitutive Translocation of ArrestinGFP Induced by Expression ofGRK2-C20

U2OS cells stably expressing arrestinGFP and the angiotensin II type 1Areceptor (AT1AR) were transiently transduced with human GRK2-C20 using abaculovirus expression system. A range of GRK2-C20 expression levels wasobtained by using varying amounts (serial dilution) of the GRK2-C20baculovirus. As shown by the black bars in FIG. 8, addition of GRK2-C20promotes ligand-independent translocation of arrestinGFP to the AT1AR. Amaximum arrestinGFP translocation response of 305±25 Fgrains and aminimum of 77±13 Fgrains was achieved with the 1:4 and 1:256 dilutions,respectively, of GRK2-C20 baculovirus. In the absence of added GRK2-C20(none), no translocation was observed (18±3 Fgrains).

To test whether the ligand-independent arrestinGFP translocation couldbe blocked by an antagonist/inverse agonist of the AT1AR, the cells weretreated with losartan. Losartan is a nonpeptide molecule that functionsas an antagonist/inverse agonist of the AT1AR. Losartan (1 μM finalconcentration) was added to the cells either immediately aftertransduction for an 18 hour incubation or 15 hours after transductionfor a 3 hour incubation. At the end of the incubation, the cells wereanalyzed for arrestinGFP translocation using the INCell Analyzer system.

As shown in FIGS. 8, 9, and 10, losartan treatment blocks theligand-independent translocation of arrestinGFP to the AT1AR induced byexpression of GRK2-C20. The most dramatic effect was observed after an18 hour incubation with losartan (FIGS. 8 and 10). At the lower levelsof GRK2-C20 expression (1:64, 1:128, and 1:256 dilutions), thistreatment paradigm results in the inhibition of greater than 90% of theligand-independent translocation of arrestinGFP to the AT1AR.

While the invention has been described and illustrated herein byreferences to various specific material, procedures and examples, it isunderstood that the invention is not restricted to the particularmaterial combinations of material, and procedures selected for thatpurpose. Numerous variations of such details can be implied as will beappreciated by those skilled in the art.

The following is a list of documents related to the above disclosure andparticularly to the experimental procedures and discussions. Thefollowing documents, as well as any documents referenced in theforegoing text, should be considered as incorporated by reference intheir entirety.

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1-13. (canceled)
 14. A method of identifying a compound that alters GPCRphosphorylation, comprising: (a) providing a cell comprising a GPCR anda GRK; (b) exposing said cell to the compound(s); and (c) determiningwhether GRK phosphorylation of the GPCR is altered in the presence ofthe compound(s).
 15. The method of claim 14, wherein the cellulardistribution of the GPCR or GRK is determined.
 16. The method of claim14, wherein a difference between (1) the distribution of the GPCR or GRKin the cell in the presence of the compound and (2) the distribution ofthe GPCR or GRK in the cell in the absence of the compound(s) ismonitored.
 17. The method of claim 16, wherein a difference iscorrelated between (1) and (2) to the phosphorylation of the GPCR. 18.The method of claim 14, wherein the GRK is not located in the plasmamembrane, indicating that GRK phosphorylation of the GPCR is altered.19. The method of claim 14, wherein the phosphorylation state of theGPCR is determined.
 20. The method of claim 14, wherein the activity ofthe GRK is determined.
 21. The method of claim 17, wherein the abilityof the GPCR to be internalized is determined.
 22. A method ofdetermining if a GPCR of interest is expressed at the plasma membrane,comprising: (a) providing a cell comprising a GPCR, an arrestin, and aGRK, wherein the arrestin is detectably labeled; (b) determining thecellular distribution of the arrestin; and (c) correlating the cellulardistribution of the arrestin to the ability of the GPCR to be expressedat the plasma membrane.
 23. The method of claim 22, wherein the arrestinis localized in vesicles, pits, endosomes, or elsewhere in thedesensitization pathway.
 24. A method of determining if a GPCR ofinterest is expressed at the plasma membrane, comprising: (a) providinga cell comprising a GPCR and a GRK, wherein the GRK is detectablylabeled; (b) determining the cellular distribution of the GRK; and (c)correlating the cellular distribution of the GRK to the ability of theGPCR to be expressed at the plasma membrane.
 25. The method of claim 24,wherein the GRK is localized at the plasma membrane.
 26. A method ofanalyzing the ability of a GPCR to bind arrestin, comprising: (a)providing a cell comprising a GPCR, an arrestin, and a GRK, wherein thearrestin is detectably labeled; (b) determining the cellulardistribution of the arrestin; and (c) correlating the cellulardistribution of the arrestin to the ability of the GPCR to bindarrestin.
 27. The method of claim 26, wherein the arrestin or the GPCRis localized in vesicles, pits, or endosomes.
 28. A compound identifiedby claim
 1. 29-36. (canceled)
 37. A modified GPCR comprising a NPXXYmotif and a carboxyl terminal tail, wherein said carboxyl terminal tailcomprises a putative site of palmitoylation and one or more clusters ofphosphorylation, wherein the carboxyl terminal tail comprises a retainedportion of a carboxyl-terminus region of a first GPCR portion fused to aportion of a carboxyl-terminus from a second GPCR, and wherein thesecond GPCR comprises the one or more clusters of phosphorylation andfurther comprises a second putative site of palmitoylation approximately10 to 25 amino acid residues downstream of a second NPXXY motif.
 38. Themodified GPCR of claim 37, wherein the first GPCR is a Class A receptor.39. The modified GPCR of claim 37, wherein the first GPCR is hGPR3,hGPR6, hGPR12, hSREB2, hSREB3, hGPR8, or hGPR22.
 40. The modified GPCRof claim 37, wherein the second GPCR is a Class B receptor.
 41. Themodified GPCR of claim 37, wherein the Class B receptor is selected fromthe group consisting of a vasopressin V2 receptor, a neurotensin-1receptor, a substance P receptor and an oxytocin receptor.
 42. A nucleicacid encoding a modified GPCR of claim
 37. 43. A nucleic acid selectedfrom the group consisting of SEQ ID Nos: 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, and
 90. 44. An expression vector comprising the nucleic acid ofclaim
 42. 45. A host cell comprising the expression vector of claim 44.46. A host cell comprising the nucleic acid of claim
 42. 47. A method ofscreening compounds for GPCR activity comprising the steps of: (a)providing a cell that expresses at least one modified GPCR according toclaim 37, wherein said cell further comprises arrestin conjugated to adetectable molecule; (b) exposing the cell to the compound; (c)detecting location of the arrestin within the cell; (d) comparing thelocation of the arrestin within the cell in the presence of the compoundto the location of the arrestin within the cell in the absence of thecompound; and (e) correlating a difference between (1) the location ofthe arrestin within the cell in the presence of the compound and (2) thelocation of the arrestin within the cell in the absence of the compound.48. The method of claim 47, wherein the arrestin is detected inendosomes, endocytic vesicles, or pits.
 49. A kit for identifying amolecule that modulates the activity of a GPCR, comprising a cell thatexpresses at least one modified GPCR according to claim 37, wherein saidcell further comprises a molecule involved in desensitization conjugatedto a detectable molecule.
 50. A method of inhibiting arrestintranslocation to the AT1AR receptor by administering an effective amountof losartan to a patient in need thereof.
 51. (canceled)
 52. The methodof claim 29, wherein the GPCR is AT1AR.
 53. The method of claim 29,wherein the disease is renal disease, diabetes and nephropathy, diabetesmellitus, type 2 diabetes, nephropathy, hypertension, congestive heartfailure, endothelial dysfunction, vascular inflammation, or heartdisease.